Compounds active in spinigosine 1-phosphate signaling

ABSTRACT

The present invention relates to S1P analogs that have activity as S1P receptor modulating agents and the use of such compounds to treat diseases associated with inappropriate S1P receptor activity. The compounds have the general structure of Formula (I) wherein R 11  is C 5 -C 18  alkyl or C 5 -C 18  alkenyl; Q is selected from the group consisting of C 3 -C 6  optionally substituted cycloalkyl, C 3 -C 6  optionally substituted heterocyclic, C 3 -C 6  optionally substituted aryl C 3 -C 6  optionally substituted heteroaryl and —NH(CO)—; R 2  is selected from the group consisting of H, C 1 -C 4  alkyl, (C 1 -C 4  alkyl)OH and (C 1 -C 4  alkyl)NH 2 ; R 23  is H or C 1 -C 4  alkyl, and R 15  is selected from the group consisting of hydroxy, phosphonate, and of Formula (II) wherein X and R 15  is selected from the group consisting of O and S; or a pharmaceutically acceptable salt or tautomer thereof.

RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) to US ProvisionalApplication Ser. Nos. 60/399,545, filed Jul. 30, 2002 and 60/425,595,filed Nov. 12, 2002, the disclosures of which are incorporated herein byreference.

US Government Rights

This invention was made with United States Government support underGrant No. NIH R01 GM52722 and NIH R01 CA88994 awarded by NationalInstitutes of Health. The United States Government has certain rights inthe invention.

BACKGROUND

Sphingosine-1-phosphate (S1P) has been demonstrated to induce manycellular effects, including those that result in platelet aggregation,cell proliferation, cell morphology, tumor-cell invasion, endothelialcell chemotaxis and endothelial cell in vitro angiogenesis. For thesereasons, S1P receptors are good targets for therapeutic applicationssuch as wound healing and tumor growth inhibition.Sphingosine-1-phosphate signals cells in part via a set of Gprotein-coupled receptors named S1P1, S1P2, S1P3, S1P4, and S1P5(formerly Edg-1, Edg-5, Edg-3, Edg-6, and Edg-8, respectively). Thesereceptors share 50-55% identical amino acids and cluster with threeother receptors (LPA1, LPA2, and LPA3 (formerly Edg-2, Edg-4 and Edg-7))for the structurally-related lysophosphatidic acid (LPA).

A conformational shift is induced in the G-Protein Coupled Receptor(GPCR) when the ligand binds to that receptor, causing GDP to bereplaced by GTP on the α-subunit of the associated G-proteins andsubsequent release of the G-proteins into the cytoplasm. The α-subunitthen dissociates from the βγ-subunit and each subunit can then associatewith effector proteins, which activate second messengers leading to acellular response. Eventually the GTP on the G-proteins is hydrolyzed toGDP and the subunits of the G-proteins reassociate with each other andthen with the receptor. Amplification plays a major role in the generalGPCR pathway. The binding of one ligand to one receptor leads to theactivation of many G-proteins, each capable of associating with manyeffector proteins leading to an amplified cellular response.

S1P receptors make good drug targets because individual receptors areboth tissue and response specific. Tissue specificity of the S1Preceptors is desirable because development of an agonist or antagonistselective for one receptor localizes the cellular response to tissuescontaining that receptor, limiting unwanted side effects. Responsespecificity of the S1P receptors is also of importance because it allowsfor the development of agonists or antagonists that initiate or suppresscertain cellular responses without affecting other responses. Forexample, the response specificity of the S1P receptors could allow foran S1P mimetic that initiates platelet aggregation without affectingcell morphology.

Sphingosine-1-phosphate is formed as a metabolite of sphingosine in itsreaction with sphingosine kinase and is stored in abundance in theaggregates of platelets where high levels of sphingosine kinase existand sphingosine lyase is lacking. S1P is released during plateletaggregation, accumulates in serum and is also found in malignantascites. Biodegradation of S1P most likely proceeds via hydrolysis byectophosphohydrolases, specifically the sphingosine 1-phosphatephosphohydrolases.

The physiologic implications of stimulating individual S1P receptors arelargely unknown due in part to a lack of receptor type selectiveligands. Therefore there is a need for compounds that have strongaffinity and high selectivity for S1P receptor subtypes. Isolation andcharacterization of S1P analogs that have potent agonist or antagonistactivity for S1P receptors has been limited due to the complication ofsynthesis derived from the lack of solubility of S1P analogs. Thepresent invention is directed to a series of compounds that are activeat S1P receptors.

SUMMARY OF VARIOUS EMBODIMENTS OF THE INVENTION

One embodiment of the present invention is directed to novelsphingosine-1-phosphate analogs, compositions comprising such analogs,and methods of using such analogs as agonist or antagonists ofsphingosine-1-phosphate receptor activity to treat a wide variety ofhuman disorders. S1P analogs of the present invention have a range ofactivities including agonism, with various degrees of selectivity atindividual S1P receptor subtypes, as well as compounds with antagonistactivity at the S1P receptors. More particularly, the S1P analogs of thepresent invention include compounds with the general structure:

-   wherein Q is selected from the group consisting of C₃-C₆ optionally    substituted cycloalkyl, C₃-C₆ optionally substituted heterocyclic,    C₃-C₆ optionally substituted aryl, C₃-C₆ optionally substituted    heteroaryl and-   R₁ is selected from the group consisting of alkyl, alkenyl, alkynyl,    alkyl(optionally substituted aryl), arylalkyl and    axylalkyl(optionally substituted)aryl;

R₁₇ is H, alkyl, alkylaryl or alkyl(optionally substituted aryl);

R₁₈ is N, O, S, CH or together with R₁₇ form a carbonyl group or a bond;

W is NH, CH₂ or (CH₂)_(n)NH (CO);

R₂ and R₃ are independently selected from the group consisting of H,NH₂, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂, with theproviso that R₂ and R₃ are not the same and either R₂ or R₃ is NH₂.

R₂₃ is selected from the group consisting of H, F, NH₂, OH, CO₂H, C₁-C₆alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;

R₂₄ is selected from the group consisting of H, F, CO₂H, OH and PO₃H₂,or R₂₃ together with R₂₄ and the carbon to which they are attached forma carbonyl group;

-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein R₁₂ is selected from the group consisting of O, NH and S;

X is selected from the group consisting of O, NH and S;

-   y is an integer ranging from 0-10; n is an integer ranging from 0-4;    and pharmaceutically acceptable salts and tautomers of such    compounds, with the proviso that R₁₈ and W are not both CH₂.    Selective agonists and antagonists at S1P receptors will be useful    therapeutically in a wide variety of human disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1F are graphic representations of [γ-35 S]GTP binding to HEK293Tcell membranes (containing different S1P receptors) in response to S1P,VPC23019 and VPC23031. FIG. 1A=S1P1 receptor, FIG. 1B=S1P3 receptor,FIG. 1C=S1P2 receptor, FIG. 1D=S1P4 receptor, FIG. 1E=S1P5 receptor, andFIG. 1F=S1P3 receptor. Each data point represents the mean of threedeterminations (CPM=counts per minute).

FIG. 2A-2E are graphic representations of [γ-35 S]GTP binding to HEK293Tcell membranes (containing different S1P receptors) in response to S1P,VPC23065 and VPC23069. FIG. 2A=S1P1 receptor, FIG. 2B=S1P3 receptor,FIG. 2C=S1P2 receptor, FIG. 2D=S1P4 receptor, and FIG. 2E=S1P5 receptor.Each data point represents the mean of three determinations (CPM=countsper minute).

FIG. 3A-3E are graphic representations of [γ-35 S]GTP binding to HEK293Tcell membranes (containing different S1P receptors) in response to S1P,VPC23075 and VPC23079. FIG. 3A=S1P1 receptor, FIG. 3B=S1P3 receptor,FIG. 3C=S1P2 receptor, FIG. 3D=S1P4 receptor, and FIG. 3E=S1P5 receptor.Each data point represents the mean of three determinations (CPM=countsper minute).

FIG. 4A-4E are graphic representations of [γ-35 S]GTP binding toHBEK293T cell membranes (containing different S1P receptors) in responseto S1P, VPC23087 and VPC23089. FIG. 4A=S1P1 recepter, FIG. 4B=S1P3receptor, FIG. 4C=S1P2 receptor, FIG. 4D=S1P4 receptor, and FIG. 4E=S1P5receptor. Each data point represents the mean of three determinations(CPM=counts per minute).

FIG. 5A and 5B. FIG. 5A is a graphic representation of [γ-35 S]GTPbinding to HEK293T cell membranes containing the S1P1 receptor, inresponse to S1P, VPC23087 and VPC23087+S1P. FIG. 5B is a graphicrepresentation of [γ-35 S]GTP binding to HEK293T cell membranescontaining the S1P3 receptor, in response to S1P, VPC23089 andVPC23089+S1P. Each data point represents the mean of threedeterminations (CPM=counts per minute).

FIGS. 6A-6D are graphic representations of [γ-35 S]GTP binding toHEK293T cell membranes (containing different S1P receptors) in responseto S1P, VPC24289 and VPC24287. FIG. 6A=S1P1 receptor, FIG. 6B=S1P3receptor, FIG. 6C=S1P4 receptor, and FIG. 6D=S1P5 receptor. Each datapoint represents the mean of three determinations, wherein the activityof VPC24289 and VPC24287 is measured relative to S1P activity at thespecific receptor subtype.

DETAILED DESCRIPTION OF EMBODIMENTS

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms.

As used herein, an “effective amount” means an amount sufficient toproduce a selected effect For example, an effective amount of an S1Preceptor antagonist is an amount that decreases the cell signalingactivity of the S1P receptor.

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo.

The term “haloalkyl” as used herein refers to an alkyl radical bearingat least one halogen substituent, for example, chloromethyl, fluoroethylor trifluoromethyl and the like.

The term “C₁-C_(n) alkyl” wherein n is an integer, as used herein,represents a branched or linear alkyl group having from one to thespecified number of carbon atoms. Typically C₁-C₆ alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The term “C₂-C_(n) alkenyl” wherein n is an integer, as used herein,represents an olefinically unsaturated branched or linear group havingfrom 2 to the specified number of carbon atoms and at least one doublebond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,and the like.

The term “C₂-C_(n) alkynyl” wherein n is an integer refers to anunsaturated branched or linear group having from 2 to the specifiednumber of carbon atoms and at least one triple bond. Examples of suchgroups include, but are not limited to, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 1-pentynyl, and the like.

The term “C₃-C_(n) cycloalkyl” wherein n=8, represents cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein, the term “optionally substituted” refers to from zero tofour substituents, wherein the substituents are each independentlyselected. Each of the independently selected substituents may be thesame or different than other substituents.

As used herein the term “aryl” refers to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, andthe like. “Optionally substituted aryl” includes aryl compounds havingfrom zero to four substituents, and “substituted aryl” includes arylcompounds having one to three substituents, wherein the substituents,including alkyl, halo or amino substituents.

The term (C₅-C₈ alkyl)aryl refers to any aryl group which is attached tothe parent moiety via the alkyl group.

The term “heterocyclic group” refers to a mono- or bicyclic arbocyclicring system containing from one to three heteroatoms wherein theeteroatoms are selected from the group consisting of oxygen, sulfur, andnitrogen.

As used herein the term “heteroaryl” refers to a mono- or bicycliccarbocyclic ring system having one or two aromatic rings containing fromone to three heteroatoms and includes, but is not limited to, furyl,thienyl, pyridyl and the like.

The term “bicyclic” represents either an unsaturated or saturated stable7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The term “lower alkyl” as used herein refers to branched or straightchain alkyl groups comprising one to eight carbon atoms, includingmethyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl and thelike.

The terms 16:0, 18:0, 18:1, 20:4 or 22:6 hydrocarbon refers to abranched or straight alkyl or alkenyl group, wherein the first integerrepresents the total number of carbons in the group and the secondinteger represent the number of double bonds in the group.

As used herein, an “S1P modulating agent” refers a compound orcomposition that is capable of inducing a detectable change in S1Preceptor activity ill vivo or in vitro (e.g., at least 10% increase ordecrease in S1P activity as measured by a given assay such as thebioassay described in Example 2).

As used herein, the term “EC₅₀ of an agent” refers to that concentrationof an agent at which a given activity, including binding of sphingosineor other ligand of an S1P receptor and/or a functional activity of a S1Preceptor (eg., a signaling activity), is 50% maximal for that S1Preceptor. Stated differently, the EC₅₀ is the concentration of agentthat gives 50% activation, when 100% activation is set at the amount ofactivity of the S1P receptor which does not increase with the additionof more ligand/agonist and 0% is set at the amount of activity in theassay in the absence of added ligand/agonist.

As used herein, the term “phosphate analog” and “phosphonate analog”comprise analogs of phosphate and phosphonate wherein the phosphorousatom is in the +5 oxidation state and one or more of the oxygen atoms isreplaced with non-oxygen moiety, including for example, the phosphateanalogs phosphorothioate, hosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,phosphoramidate, boronophosphates, and the like, including associatedcounterions, e.g., H, NH₄, Na, and the like if such counterions arepresent

The S1P analogs of the present invention contain one or more asymmetriccenters in the molecule. In accordance with the present invention astructure that does not designate the stereochemistry is to beunderstood as embracing all the various optical isomers, as well asracemic mixtures thereof.

The compounds of the present invention may exist in tautomeric forms andthe invention includes both mixtures and separate individual tautomers.For example the following structure:

is understood to represent a mixture of the structures:

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the S1P analogs of thepresent invention and which are not biologically or otherwiseundesirable. In many cases, the S1P analogs of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenylarnines, di(substituted alkenyl) amines, tri(substituted alkenyl)amines, cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl)amines, substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstitutedcycloalkenyl amines,atyl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteraryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

Embodiments

One aspect of the present invention is directed to novel S1P analogsthat have activity as modulators of S1P receptor activity. Modulators ofS1P activity include agents that have either agonist or antagonistactivity at the S1P receptor as well as analogs of those compounds thathave been modified to resist enzymatic modification (i.e. blockmodification of the compounds by phosphohydrolases, sphingosine lyasesor sphingosine kinases), or provide a suitable substrate for sphingosinekinases to convert an administered form into a more active form.

The structure of S1P can be described as a combination of three regions:the phosphate head group, the linker region, and the fatty acid tail.Through structure activity relationships (SAR) of the closely relatedlysophospholipid, lysophosphatidic acid (LPA), it has been determinedthat the presence of a phosphate head group is an important feature toallow binding of S1P to its S1P receptors. However, there are exceptionsto the requirement for a phosphate head group. In particular aphosphonate, hydroxyl, phosphate or phosphonate group can be substitutedfor the phosphate head group while retaining activity at the S1Preceptor.

Based on the SAR of LPA, the linker region of S1P is anticipated to bethe area of the molecule that can best accommodate change. Again usingthe SAR of LPA as a lead, it is presumed that presence of a hydrogenbond donor 5 bonds away from the phosphate is important to binding. Froma retrosynthetic standpoint, the linker region may be seen as afunctionalized derivative of L-Serine.

Due to the long fatty acid chain and charged phosphate head group, S1Phas an amphipathic nature that makes it extremely insoluble in organicsolvents. Manipulation of the saturation of the fatty acid chain maycompromise aggregate formation of the molecule, thereby increasingsolubility. One important aspect of the long chain, however, is thelength. GTP_(γ)S studies that have been completed thus far havedemonstrated that an 18 carbon backbone, as is the case in S1P, displaysoptimal activity compared to 16 and 20 carbon backbones, however thelong fatty acid chain can vary from 8 to 25 carbons and still exhibitactivity.

It is also anticipaed that the S stereochemistry of the C-2amine mayhave an effect on binding as one would expect from a receptor. Hydrogenbonds from the phosphate head group and the C-2 amine to adjacentargenine and glutamic acid residues on the model receptor have beendemonstrated to be important to S1P-receptor binding. In accordance withone embodiment an S1P receptor modulating compound is provided whereinthe compound has the general structure:

-   wherein-   W is CR₂₇R₂8 or (CH₂),NH (CO);

wherein R₂₇ and R₂₈ are independently selected from the group consistingof H, halo and hydroxy;

-   Y is selected from the group consisting of a bond, CR₉R₁₀, carbonyl,    NH, O or S;

wherein R₉ and R₁₀ are independently selected from the group consistingof H, halo, hydroxy and amino;

-   Z is CH₂, aryl, flourine substituted aryl or heteroaryl;-   R₁₁ and R₁₆ are independently selected from the group consisting of    C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, C₅-CIs alkoxy,    (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀ heteroaryl)R₂₀, C₅-C₁₀    (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀    alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀;

wherein R₂₀ is or C₁-C₁₀ alkyl;

-   R₂₉ is H or halo;-   R₁₇ is selected from the group consisting of H, halo, NH₂, C₁-C₆    alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₁-C₆ alkylcyano and C₁-C₆    alkylthio;-   R₂, and R₂₁ are both NH₂;-   R₃ is selected from the group consisting of H, C₁-C₆ alkyl, (C₁-C₄    alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)aryl(C₀-C₄ alkyl) and    (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄ alkyl);-   R₂₂ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄    alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)aryl(C₀-C₄ alkyl) and    (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄ alkyl);-   R₂₃ is selected from the group consisting of H, F, NH₂, OH, CO₂H,    C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;-   R₂₄ is selected from the group consisting of H, F, CO₂H, OH and    PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they are    attached form a carbonyl group;-   R₂₅, R₇ and R₈ are independently selected from the group consisting    of O, S, CHR₂₆, CR₂₆, NR₂₆, and N;

wherein R₂₆ is H or C₁-C₄ alkyl;

-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein R₁₂ is selected from the group consisting of O, NH and S;

X is selected from the group consisting of O, NH and S;

-   y and m are integers independently ranging from 0 to 4;-   p and q are integers independently ranging from 1 to 10;-   n is an integer ranging from 0 to 10;    or a pharmaceutically acceptable salt or tautomer thereof with the    proviso that W and Y are not both methylene.

In one embodiment the present invention is directed to a S1P receptormodulating compound is represented by the formula:

wherein

-   -   Z is CH₂, aryl or heteroaryl;    -   R₁₆ is selected from the group consisting of C₁-C₁₈ alkyl,        C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, C₅-C₁₈ alkoxy,        (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀ (heteroaryl)R₂₀,        C₅-C₁₀ (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C_(s)-C₁₀        alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀, wherein        R₂₀ is H or C₁-C₁₀ alkyl;

-   R₁₇ is selected from the group consisting of H, halo, NH₂, C₁-C₆    alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₁-C₆ alkylcyano and C₁-C₆    alkylthio;

-   R₂₁ is selected from the group consisting of NH₂, OH, C₁-C₆ alkyl,    (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)aryl(C₀-C₄ alkyl)    and (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄ alkyl), with the proviso that R₂    or R₃ is NH₂;

-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein X and R₁₂ are independently selected from the group consistingof O and S;

-   R₂₃ is selected from the group consisting of H, F, NH₂, OH, CO₂H,    C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;-   R₂₄ is selected from the group consisting of H, F, CO_(z)H, OH and    PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they are    attached form a carbonyl group; p and q are integers independently    ranging from 1 to 10;-   y is an integer ranging from 0 to 4; and-   n is an integer ranging from 0 to 10;    or a pharmaceutically acceptable salt or tautomer thereof In one    embodiment the compound of Formula II is provided wherein Z is CH₂,    y is 0, n is 1-10, and R₁₇ is H. In another embodiment, the compound    of Formula 1 is provided wherein Z is C₅-C₆ aryl or C₅-C₆    heteroaryl, y is 0, n is 0, R₁₇ and R₂₃ are each H and R₁₆ is    selected from the group consisting of C₅-C₁₂ alkyl, C₂-C₁₂ alkenyl    or C₅-C₁₂ alkoxy. In another embodiment, the compound of Formula II    is provided wherein Z is C₅-C₆ aryl or C₅-C₆ heteroaryl, y is 0, n    is 0, R₁₇, R₂₃ and R₂₄ are each H, R₁₆ is selected from the group    consisting of C₅-C₁₂ alkyl, C₂-C₁₂ alkenyl or C₅-C₁₂ alkoxy and R₁₅    is hydroxy.

In another embodiment of the present invention a S1P receptor modulatingcompound is provided wherein the compound is represented by the formula:

-   wherein Z is aryl or heteroaryl;-   R₁₆ is selected from the group consisting of C₅-C₁₈ alkyl, C₅-C₁₈    alkenyl, C₅-C₁₈ is alkynyl and C₅-C₁₈ alkoxy;-   Y is selected from the group consisting of CHOH, CF₂, CFH, carbonyl,    NH, O and S;

W is CR₂₇R₂₈, wherein R₂₇ and R₂₈ are independently selected from thegroup consisting of H, halo and hydroxy;

-   R₂₁ is selected from the group consisting of NH₂, OH, C₁-C₆ alkyl,    (C₁-C₄ alkyl)OH, (C₁-C₄ aklyl)NH₂, (C₁-C₄ alkyl)aryl(C₀-C₄ alkyl)    and (C₁-C₄ alkyl)aryloxyaUyl(C₀-C₄ alkyl);-   R₂₃ is selected from the group consisting of H, F, NH₂, OH, CO₂H,    C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;-   R₂₄ is selected from the group consisting of H, F, CO₂H, OH and    PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they are    attached form a carbonyl group;-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein X and R₁₂ are independently selected from the group consistingof O and S; and y is an integer ranging from 0 to 4; or apharmaceutically acceptable salt or tautomer thereof. In one embodimentthe compound of Formula m is provided wherein Z is C₅-C₆ aryl or C₅-C₆heteroaryl, R₂₃ and R₂₄ are both H, R₂₁ is selected from the groupconsisting of OH, C₁-C₄ alkyl, and (C₁-C₃ alkyl)OH; and y is 0.

In another embodiment, the compound is represented by the formula:

-   wherein R₁₆ is selected from the group consisting of C₅-C₁₂ aUkyl,    C₅-C₁₂ alkenyl and C₅-C₁₂ alkynyl;-   Y is selected from the group consisting of carbonyl, NH and O;-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein X and R₁₂ are independently selected from the group consistingof O and S;

-   R₂₁ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄    alkyl)OH and (C₁-C₄ alkyl)NH₂; R₂₃ and R₂₄ are independently    selected from the group consisting of H, OH, F, CO₂H or PO₃H₂ or R₂₃    together with R₂₄ and the carbon to which they are attached form a    carbonyl group, as well as pharmaceutically acceptable salts and    tautomers thereof.

In another embodiment, the compound of Formula m is provided whereinwherein Z is C₅-C₆ aryl;

-   R₁₆ is selected from the group consisting of C₅-C₁₈ alkyl and C₅-C₁₈    alkenyl;-   Y is selected from the group consisting of CF₂, CFH, carbonyl, NH, O    and S;-   W is CH₂;-   R₂, is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄    alkyl)OH and (C₁-C₄ alkyl)NH₂;-   R₂₃ and R₂₄ are both H; y is 0; and-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein R₁₂ is O and S (and in one embodiment R₁₅ is OH),

or a pharmaceutically acceptable salt or tautomer thereof.

In another embodiment of the present invention a S1P receptor modulatingcompound is provided wherein the compound is represented by the formula:

wherein

-   R₁₁ is selected from the group consisting of C₅-C₁₂ allyl, C₅-C₁₂    alkenyl and C₅-C₁₂ allynyl;-   R₂₉, is H or halo;-   R₂₅, R₇ and R₈ are independently selected from the group consisting    of O, S, CHR₂₆, CR₂₆, NR₂₆, and N;

wherein R₂₆ is H, F or C₁-C₄ alkyl;

-   R₂, is selected from the group consisting of H, NH₂, OH, C₁-C₆    alkyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)aryl(C₀-C₄    alkyl) and (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄ alkyl);-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein X and R₁₂ are independently selected from the group onsisting ofO and S;

-   R₂₃ and R₂₄ are independently selected from the group consisting of    H, OH, F, CO₂H, C₁-C₃ alkyl or PO₃H₂ or R₂₃ together with R₂₄ and    the carbon to which they are attached form a carbonyl group;-   m is 1 or 0; and-   y is an integer ranging from 0 to 4;    or a pharmaceutically acceptable salt or tautomer thereof. In one    embodiment R₂₉ is H or F; m is 0; y is 1 or 0; R₂ is selected from    the group consisting of H, C₁-C₆ alkyl and (C₁-C₄ alkyl)OH; R₂₄ is H    and R₂₃ is C₁-C₃ alkyl. In accordance with one embodiment of the    present invention a compound of Formula IV, V or VI is provided    wherein R₂₃ and R₂₉ are both H; m is 0; R₂₅ is CH₂ or CH; R₇ and kg    are independently selected from the group consisting of O, CH₂ or    CH, NH, and N; R₂, is selected from the group consisting of H, F,    C₁-C₄ alkyl and (C₁-C₄ alkyl)OH; R₂₄ is selected from the group    consisting of H, F, C₁-C₃ alkyl; and y is 1 or 0.

In one embodiment of the present invention a S1P receptor modulatingcompound is provided wherein the compound is represented by the formula:

wherein

-   R₁₁ is selected from the group consisting of C₅-C₁₅ alkyl, C₅-C₁₅    alkenyl and C₅-C₁₅ alkynyl;-   R₇ and R₈ are independently selected from the group consisting of O,    S, NH and N;-   R₂, is selected from the group consisting of H, C₁-C₆ alkyl, (C₁-C₄    alkyl)OH, and (C₁-C₄ alkyl)NH₂;-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein X and R₁₂ are independently selected from the group onsisting ofO and S;

-   R₂₃ is selected from the group consisting of H, F and OH;-   R₂₄ is selected from the group consisting of H, F, OH and PO₃H₂, or    R₂₃ together with R₂₄ and the carbon to which they are attached form    a carbonyl group;-   m is 0; and-   y is an integer ranging from 0 to 4;    or a pharmaceutically acceptable salt or tautomer thereof.

In one embodiment of the present invention a S1P receptor modulatingcompound is provided wherein the compound is represented by the formula:

-   wherein R₁₁ is selected from the group consisting of C₅-C₁₂ alkyl,    C₅-C₁₂ alkenyl and C₅-C₁₂ alkynyl;-   R₇ and R₈ are independently selected from the group consisting of O,    S, CH₂, CH, NH and N;-   R₂ and R₃ are independently selected from the group consisting of H,    NH₂, OH, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄    akyl)aryl(C₀-C₄ alkyl) and (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄ alkyl),    with the proviso that R₂ and R₃ are not the same and either R₂ or R₃    is NH₂;-   y is 1 or 0-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein R₁₂ is selected from the group consisting of O and S;

-   R₂₃ is selected from the group consisting of H, F, CO₂H, C₁-C₄ alkyl    and OH;-   R₂₄ is selected from the group consisting of H, F, C₁-C₄ alkyl and    PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they are    attached form a carbonyl group; as well as pharmaceutically    acceptable salts or tautomers thereof.

In accordance with one embodiment of the present invention a compound ofFormula VII is provided wherein R₂₃ is H; R₂₄ is selected from the groupconsisting of H, F, C₁-C₄ alkyl; and R₇ and R₈ are independentlyselected from the group consisting of O, NH and N. In anotherembodiment, a compound of Formula VII is provided wherein R₂₃ is H; R₂is NH₂; and R₃ is selected from the group consisting of H, C₁-C₄ alkyl,(C₁-C₄ alkyl)OH and (C₁-C₄ alkyl)NH₂. Alternatively, in one embodiment acompound of Formula VII is provided wherein R₂₃ is H; R₃ is NH₂; and R₂is selected from the group consisting of H, C₁-C₄ alkyl, (C₁-C₄ allyl)OHand (C₁-C₄ aIyl)NH₂. In another embodiment, a compound of Formula VII isprovided wherein R₂₃ is H; R₂ is NH₂; and R₃ is selected from the groupconsisting of H, C₁-C₄ alkyl, (C₁-C₄ alkyl)OH and (C₁-C₄ alkyl)NH₂; R₂₄is selected from the group consisting of H, F, C₁-C₄ alkyl; and R₇ andR₈ are independently selected from the group consisting of O, NH and N.In another embodiment a compound of Formula VII is provided wherein R₁₁is selected from the group consisting of C₅-C₁₂ alkyl or C₅-C₁₂ alkenyl;R₇ and R₈ are independently selected from the group consisting of O, NHand N; R₂ and R₃ are independently selected from the group consisting ofH, NH₂, C₁-C₆ alkyl and (C₁-C₄ alkyl)OH, with the proviso that R₂ and R₃are not the same and either R₂ or R₃ is NH₂; y is 0; R₁₅ is hydroxy; R₂₃is H; and R₂₄ is H, F or C₁-C₄ alkyl; as well as pharmaceuticallyacceptable salts or tautomers thereof.

In one embodiment of the present invention a S1P receptor modulatingcompound is provided wherein the compound is represented by the formula:

-   wherein R₁₁ is selected from the group consisting of C₅-C₁₂ allyl,    C₅-C₁₂ alkenyl and C₅-C₁₂ alkynyl;-   R is O or N;-   R₂ and R₃ are independently selected from the group consisting of    NH₂, C₁-C₆ alkyl and (C₁-C₄ alkyl)OH, with the proviso that R₂ and    R₃ are not the same and either R₂ or R₃ is NH₂;-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein R₁₂ is selected from the group consisting of O and S;

-   R₂₃ is H or F; and-   R₂₄ is H, F or C₁-C₄ alkyl; as well as pharmaceutically acceptable    salts or tautomers thereof. In one embodiment the compound of    Formula VIII is provided wherein R₁ is C₅-C₁₂ alkyl or C₅-C₁₂    alkenyl; R₈ is N; R₂ and R₃ are independently selected from the    group consisting of NH₂, CH₃ and (C₁-C₃ alkyl)OH, with the proviso    that R₂ and R₃ are not the same and either R₂ or R₃ is NH₂; and R₁₅    is hydroxy; R₂₃ is H; and R₂₄ is H or C₁-C₄ alkyl as well as    pharmaceutically acceptable salts or tautomers thereof. In another    embodiment the compound of Formula VIII is provided wherein R₁ is    C₅-C₁₂ alkyl or C₅-C₁₂ alkenyl; R₈ is N; R₂ and R₃ are independently    selected from the group consisting of NH₂, CH₃ and (C₁-C₃ allyl)OH,    with the proviso that R₂ and R₃ are not the same and either R₂ or R₃    is NH₂; and R₁₅ is hydroxy; R₂₃ is H; and R₂₄ is H or CH₃ as well as    pharmaceutically acceptable salts or tautomers thereof.

In one embodiment a S1P receptor modulating compound is provided whereinthe compound is represented by the formula:

-   wherein R₁ is C₅-C₁₈ allky or alkenyl;-   R₈ is N-   R₂ is NH₂;-   R₃ is CH₃ or (C₁-C₃ alkyl)OH and R₁₅ is hydroxy;    or a pharmaceutically acceptable salt or tautomer thereof

In accordance with one embodiment an S1P receptor modulating compound isprovided wherein the compound has the general structure:

wherein R₁ is selected from the group consisting of alkyl, alkenyl,alkynyl, alkyl(optionally substituted aryl), alkyl(optioraliysubstituted cycloalkyl), arylalkyl, and arylalkl (optionallysubstituted) aryl;

R₁₂ is O, or R₁ and R₁₂ taken together form an optionally substitutedheteroaryl;

-   R₁₇ is H, C₁-C₄ allkl or (CH₂)aryl;-   R₂ and R₃ are independently selected from the group consisting of H,    NH₂, OH, C₁-C₆ alkyl, —(C₁-C₄ alkyl)OH, and —(C₁-C₄ alkyl)NH₂; y is    an integer from 1-10, and R₄ is selected from the group consisting    of hydroxyl, phosphate, methylene phosphonate, α-substituted    methylene phosphonate, phosphate anlogs and phosphonate analogs or a    pharmaceutically acceptable salt thereof. In one embodiment one of    the R₂ and R₃ substituents of Formula XII is NH₂.Examples of    pharmaceutically acceptable salts of the compounds of the Formula    XII include salts with inorganic acids, such as hydrochloride,    hydrobromide and sulfate, salts with organic acids, such as acetate,    fimarate, maleate, benzoate, citrate, malate, methanesulfonate and    benzenesulfonate salts, and when a carboxy group is present, salts    with metals such as sodium, potassium, calcium and aluminium, salts    with amines, such as triethylamine and salts with dibasic amino    acids, such as lysine. The compounds and salts of the present    invention encompass hydrate and solvate forms.

In one embodiment, an S1P modulating compound is provided having thegeneral structure:

-   wherein R₁ is selected from the group consisting of C₈-C₂₂ alkyl,    C₈-C₂₂ alkenyl, C₈-C₂₂ alkyl and —(CH₂)_(n)—Z—R₆;-   R₅ is selected from the group consisting of hydroxyl, phosphonate,    α-substituted methylene phosphonate, phosphate analogs and    phosphonate analogs;-   y is an integer ranging from 1 to 4;-   n is an integer ranging from 0 to 10;-   Z is selected from the group consisting of cycloalkyl, aryl and    heteroaryl; and-   R₆ is selected from the group consisting of H, C₁-C₁₂ alyl, C₁-C₂₀    alkoxy, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino or a    pharmaceutically acceptable salt thereof. When R₈ is an alpha    substituted phosphonate, the alpha carbon can be mono- or di-    substituted, wherein the substitutions are independently selected    from the group consisting of H, OH, F, CO₂H, PO₃H₂, or together with    the attached carbon, form a carbonyl. In one embodiment, R₁ is    C₈-C₂₂ alkyl, and more preferably C₁₂-C₁₆ alkyl, y is 1 or 2 and R₅    is hydroxy, phosphate or phosphonate. Alternatively, in one    embodiment, R₁ is —(CH₂)_(n)—Z—R₆, wherein n is an integer ranging    from 14, Z is aryl and R₆ is C₁-C₁₀ alkyl; more preferably, Z is    phenyl, R₅ is hydroxy, phosphate or phosphonate, and R₆ is C₆-C₁O    alkyl.

In another embodiment of the present invention an S1P modulatingcompound is provided having the general structure:

-   wherein R₁₄ is selected from the group consisting of H, hydroxy,    NH₂, C₈-C₂₂ alkyl, C₈-C₂₂ alkenyl, C₅-C₂₂ alkynyl and —(CH₂)—Z—R₆;-   R₄ is selected from the group consisting of hydroxyl, phosphate,    phosphonate, α-substituted methylene phosphonate, phosphate analogs    and phosphonate analogs;-   y is an integer ranging from 1 to 4;-   m is an integer ranging from 0 to 4;-   n is an integer ranging from 0 to 10;-   Z is selected from the group consisting of cycloalkyl, aryl and    heteroaryl; and-   R₆ is selected from the group consisting of H, C₁-C₂ alkyl, C₁-C₂₀    alkoxy, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino; and-   R₇ and R₈ are independently selected from the group consisting of O,    S and N.

In one embodiment R₁ is selected from the group consisting of C₈-C₂₂alkyl, C₈-C₂₂ alkenyl and C₈-C₂₂ alkynyl, R₄ is hydroxyl, phosphate orphosphonate, y is 1 or 2, m is 0 or 1 and either R₇ or R₈ is N; morepreferably, R₁ is C₄-C₁₀ alkyl, R₈ is hydroxyl or phosphate, y is 1, mis 0 and R₇ and R₈ are both N.

The present invention also encompasses compounds of the generalstructure:

wherein R₉ is selected from the group consisting of —NR₁, and —OR₁;

-   R₁ is selected from the group consisting of C₈-C₂₂ alkyl and

wherein R₆ and R₁₃ are independently selected from the group consistingof H, C₁-C₁₀ alkyl and C₁-C₂₀ alkoxy and R₁₀ is hydroxy, phosphonate,methylene phosphonate or phosphate, with the proviso that when R₉ is—NR₁, R₁₀ is not phosphate. In one preferred embodiment, R₉ is —NR₁, R₆is C₁-C₁₀ alkyl, R₁₃ is H and R₁₀ is hydroxy, phosphonate, or methylenephosphonate.

A GTP[γ35 S] binding assay was developed to analyze directly theactivation of individual S1P receptors, and thus allow theidentification of S1P receptor agonists and antagonists as well asdetermine the relative efficacies and potencies at each receptor in acommon system. The same results were obtained regardless of whether therecombinant receptor used exogenous G proteins (HEK293T cells) orendogenous G proteins (RH7777 cells). In addition, insect Sf9 cellsinfected with recombinant baculovirus encoding receptors (e.g. LPA andS1P receptors) and G proteins can also serve as the source of membranesfor the broken cells used in the GTPgammaS-35 binding assays. The Sf9cell and HEK293T cell membranes gave similar results. Furthermore, theactivities measured in the broken cell assay predicted the responsesseen in whole cell assays. Thus the primary assay used in the presentinvention for detemining compound potency and efficacy is a validmeasure of activity at the S1P receptors.

The GTP[y35 S] binding assay has revealed that the compounds of thepresent invention have the ability to modulate S1P receptor activity(See Examples 2 and 3). More particularly, compounds represented by thefollowing formula display activity as modulators of S1P activity. Moreparticularly, such compounds include those having the structure

-   wherein-   W is CR₂₇R₂₈ or (CH₂)_(n)NH (CO);

wherein R₂₇ and R₂₈ are independently selected from the group consistingof H, halo and hydroxy;

-   Y is selected from the group consisting of a bond, CR₉R₁₀, carbonyl,    NH, O or S;

wherein R₉ and R₁₀ are independently selected from the group consistingof H, halo, hydroxy and amino;

-   Z is CH₂, aryl, halo substituted aryl or heteroaryl;-   R₁₁ and R₁₆ are independently selected from the group consisting of    C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, C₅-C₁₈ alkoxy,    (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀ (heteroaryl)R₂₀,    C₅-C₁₀ (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀    alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀;

wherein R₂₀ is H or C₁-C₁₀ alkyl;

-   R₂₉ is H or halo;-   R₁₇ is selected from the group consisting of H, halo, NH₂, C₁-C₆    alkyl, C₁-C₆ alkoxy, C₁-C₆ allylarino, C₁-C₆ alkylcyano and C₁-C₆    alkylthio;-   R₂ and R₂₁ are both NH₂;-   R₃ is selected from the group consisting of H, C₁-C₆ alkyl, (C₁-C₄    alkyl)OH, and (C₁-C₄ alkyl)NH₂;-   R₂₂ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄    alkyl)OH and C₁-C₄ alkyl)NH₂;-   R₂₃ is selected from the group consisting of H, F, CO₂H, OH, C₁-C₆    alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ aIyl)NH₂;-   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or R₂₃    together with R₂₄ and the carbon to which they are attached form a    carbonyl group;-   R₂₅, R₇ and R₈ are independently selected from the group consisting    of O, S, CHR₂₆, CHR₂₆, NR₂₆, and N;

wherein R₂₆ is H, F or C₁-C₄ alkyl;

-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein R₁₂ is selected from the group consisting of O, NH and S;

X is selected from the group consisting of O, NH and S;

-   y and m are integers independently ranging from 0 to 4;-   p and q are integers independently ranging from 1 to 10;-   n is an integer ranging from 0 to 10;    or a pharmaceutically acceptable salt or tautomer thereof, with the    proviso that W and Y are not both methylene.

As described in Example 2 compounds having the general structure

-   wherein R₉ is selected from the group consisting of —NR₁ and —OR₁,    R₁ is C₈-C₂₂ alkyl, R₂ and R₃ are independently selected from the    group consisting of H and NH₂, wherein at least one of R₂ and R₃ is    NH₂ and R is phosphate all display significant agonist activity at    the S1P receptors tested (S1P1, S1P2, S1P3, S1P5), although none    were as potent as S1P itself (See Table 1 of Example 2). However,    one compound, VPC22135 (wherein R₂ is H, R₃ is NH₂, R₄ is phosphate    and R₉ is —N(CH₂)₁₃CH₃), approached the potency of S1P at both the    human S1P1 and human S1P3 receptors. In accordance with one    embodiment of the present invention compound VPC22135 is used as a    selective agonist of human S1P1 and human S1P3 receptors. Curiously,    this compound has the amino group in the unnatural (R)    configuration. Its enantiomer, VPC22053, was more than 1 log order    less potent at both the S1P1 and S1P3 receptors.

An additional series of compounds have shown activity in modulating S1Preceptor activity, however these compounds also displayed selectivityfor certain S1P receptor subtypes (See Example 3 and FIGS. 1-5). Each ofthese compounds (VPC 23019, 23031, 23065, 23069, 23087, 23089, 23075,23079) are inactive at the S1P2 receptor. Compounds VPC23031, 23019,23089 are inverse agonists (antagonists of the S1P3) receptor, but thisinverse agonism becomes agonism when the alkyl chain length is 9 carbons(VPC23079) or 10 (VPC23069). In accordance with one embodiment of thepresent invention an antagonist of S1P activity is provided. Inparticular, a compound having the general structure:

wherein R₁ and R₁₁ is C₄-C₁₂ alkyl and located in the meta or orthoposition,

-   Q is selected from the group consisting of C₃-C₆ optionally    substituted cycloalkyl, C₃-C₆ optionally substituted heterocyclic,    C₃-C₆ optionally substituted aryl and C₃-C₆ optionally substituted    heteroaryl;-   R₂ is selected from the group consisting of H, C₁-C₄ alkyl and    (C₁-C₄ alkyl)OH;-   R₂₃ is selected from the group consisting of H, F, CO₂H, OH, C₁-C₆    alky, (C₁-C₄ alkyl)OH, and (C₁-C₄ aIkyl)NH₂;-   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or R₂₃    together with R₂₄ and the carbon to which they are attached form a    carbonyl group; and-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and-   wherein X and R₁₂ is selected from the group consisting of O and S;-   or a pharmaceutically acceptable salt or tautomer thereof are    anticipated to have antagonist activity at the S1P3 receptor. In    accordance with one embodiment the R₁ substituent is located in the    ortho position on the phenyl ring, and in one embodiment the R₁    substituent is located in the meta position on the phenyl ring.

However compounds of the general structure

-   (wherein R₁₁ is located in the para position) have exhibited    activity as agonists of S1P activity. In particular compounds of    Formula XI are provided as S1P agonists wherein R₁ is C₅-C₁₈ alkyl    or C₅-C₁₈ alkenyl;-   Q is selected from the group consisting of C₃-C₆ optionally    substituted cycloalkyl, C₃-C₆ optionally substituted heterocyclic,    C₃-C₆ optionally substituted aryl and C₃-C₆ optionally substituted    heteroaryl;-   R₂ is selected from the group consisting of H, C₁-C₄ alkyl and    (C₁-C₄ alkyl)OH;-   R₂₃ is selected from the group consisting of H, F, CO₂H, OH, C₁-C₆    alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;-   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or R₂₃    together with R₂₄ and the carbon to which they are attached form a    carbonyl group; and-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and-   wherein X and R₁₂ are independently selected from the group    consisting of O and S;-   or a pharmaceutically acceptable salt or tautomer thereof and a    pharmaceutically acceptable carrier. In one embodiment a compound    represented by Formula XI is provided as an S1P agonist wherein-   R₁₁ is C₅-C₁₈ alkyl or C₅-C₁₈ alkenyl;    Q is selected from the group consisting of —NH(CO)—,-   R₂ is selected from the group consisting of H, C₁-C₄ alkyl and    (C₁-C₄ alkyl)OH;-   R₂₄ is H;-   R₂₃ is H or C₁-C₄ alkyl, and-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and-   wherein X and R₁₂ are independently selected from the group    consisting of O and S.

Compounds VPC23065, VPC23087 and VPC23075 are primary alcohols, i.e. R₄formula XII is hydoxy. These compounds demonstrate significant agonistactivity at various S1P receptors. In particular, the S1P4 receptorbinds to the primary alcohol S1P analogs with an EC₅₀ within a log orderof the phosphorylated compounds. Since S1P4 is present on lymphocytes,the use of the primary alcohol analogs may be used forimmuno-suppression. In addition, it is also hypothesized that thehydroxy moiety of the primary alcohols may be converted to phosphates invivo. Therefore the primary alcohol S1P analogs of the present inventionare all anticipated to serve as prodrug forms of active S1P receptormodulating compounds.

S1P is metabolized by a variety of conceivable routes includingphosphatases, esterases or transported into cells. The S1P signal atreceptors might be prolonged if the routes of degradation could beevaded or inhibited by S1P structural analogs. The S1P analogs of thepresent invention can be used, in accordance with one embodiment, toinhibit or evade endogenous S1P metabolic pathways includingphosphotases, esterases, transporters and S1P acyl transferases. Forexample those S1P analogs that lack an ester bond would be resistant todegradation by endogenous esterases. One embodiment of the presentinvention is directed to compounds that function as a S1P receptoragonists and antagonists that are resistant to hydrolysis by lipidphosphate phosphatases (LPPs) or are sub-type selective inhibitors ofLPPs, and in particular are resistant to hydrolysis by sphingosine1-phosphate phosphohydrolase. Previously described S1P mimetics containa phosphate group, and thus are likely susceptible to hydrolysis byLPPs.

Alpha hydroxy phosphonates are well known phosphate minetics. Forexample, the compounds used clinically to treat osteoporosis(pamidronate, alendronate) are alpha hydroxy bisphosphonates that areanalogs of pyrophosphate. S1P analogs can be prepared wherein thephosphate moiety is replaced by an alpha substituted phosphonate,wherein the substituents are selected from the group consisting of H,OH, F, CO₂H, PO₃H₂ or double bonded oxygen. Accordingly, one aspect ofthe present invention is directed to lipid phosphate phosphataseresistant S1P analogs having the general structures:

-   wherein R₉ is selected from the group consisting of —NR₁ and —OR₁;-   R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂    alkenyl, C₈-C₂₂ alkynyl and —(CH₂)_(n)—Z—R₆;-   R₁₁ is —(CH₂)_(n)—Z—R₆; wherein n is an integer ranging from 0 to    10, Z is selected from the group consisting of aryl and heteroaryl    and R₆ is selected from the group consisting of H, C₁-C₁₀ alkyl,    C₁-C₂₀ alkoxy, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino;-   R₂ and R₃ are independently selected from the group consisting of H,    NH₂, OH, C₁-C₆ alkyl, —(C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂, —(C₁-C₄    alkyl)aryl(C₀-C₄ alkyl) and —(C₁-C₄ alkyl)aryloxyaryl(C₀-C₄ alkyl),    wherein R₂ and R₃ are not the same and R₂ or R₃ is NH₂-   y is an integer from 0-10;-   R₁₄ is selected from the group consisting of

OH;

-   R₁₅ is selected from the group consisting of H, hydroxy, amino,    COOH, halo, PO₂H₂; or R₁₅ and R₁₆ taken together form a keto group    or a methylene group;-   R₁₆ is selected from the group consisting of hydroxy, amino, COOH,    halo, PO₂H₂; or R₁₅ and R₁₆ taken together with the carbon to which    they are bound form a carbonyl or a methylene group; and-   R₁₇ is selected from the group consisting of O, S and NH. In one    preferred embodiment, R₉ is —NR₁ wherein R₁ is C₈-C₂₂ alkyl or    —(CH₂)_(n)—Z—R₆, y is 0 or 1, R₁₅ and R₁₆ are independently H, C₁-C₄    alkyl or hydroxyl, and R₁₄ is OH. In an alternative preferred    embodiment the compound has the general structure:-   wherein R₉ is selected from the group consisting of —NR₁ and —OR₁;

R₁ is selected from the group consisting of C₈-C₂₂ alkyl, C₈-C₂₂alkenyl, C₈-C₂₂ alkynyl and —(CH₂)_(n)—Z—R₆, wherein n is an integerranging from 0 to 10, Z is selected from the group consisting of aryland heteroaryl and R₆ is selected from the group consisting of H, C₁-C₁₀alkyl, C₁-C₂₀ alkoxy, C₁-C₂₀ alkylthio, and C₁-C₂₀ alkylamino;

-   R₂ is NH₂ or OH;-   y is an integer from 0-10;-   R₁₄ is H or-   R₁₅ is NH₂ or OH; and-   R₁₇ is selected from the group consisting of O, S and NH. In one    preferred embodiment, R₉ is —NR₁, wherein R₁ is C₅-C₂₂ alkyl or    (CH₂)_(n)—Z—R₆, y is 0 or 1, and R₁₇ is O.

Lysophospholipids such as S1P and LPA, and their phosphate-containinganalogs, are probably degraded by membrane bound lipidectophosphohydrolases. This activity can be evaded by substitutingphosphonate, α-substituted phosphonate, phosphothionate or otherphosphate analogs as phosphate surrogates. Such compounds might alsofunction as lipid ectophosphohydrolase inhibitors. Further, substitutionof small alkyl groups (e.g. C₁-C₄ alkyl, C₁-C₃ alkylalcohol) at C-1 orC-2 might retard lipid ectophosphohydrolase cleavage by sterichindrance.

In accordance with one embodiment an S1P receptor modulating compound isprovided wherein the compound has the general structure:

-   wherein R₁ is selected from the group consisting of alkyl, alkenyl,    alkynyl, alkyl (optionally substituted aryl), alkyl (optionally    substituted cycloalkyl), arylalkyl and arylalkyl (optionally    substituted aryl) R₇ is H, O, or R₁ and R₇ taken together form an    optionally substituted C₃-C₆ heteroaryl or optionally substituted    C₃-C₆ heterocyclic group; R₆ is H, C₁-C₄ alkyl or (CH₂)aryl; R₂ and    R₃ are independently selected from the group consisting of H. NH₂,    OH, C₁-C₆ aiOkyl, C₁-C₄ aIkyl)OH, and —(C₁-C₄ alkyl)NH₂; R₄ and R₈    are independently selected from the group consisting of H, NH₂, OH,    C₁-C₆ alkyl, —(C₁-C₄ alkyl)OH, and —(C₁-C₄ alkyl)NH₂; R₈ is O, NH    or S. In one embodiment, one of the R₂ and R₃ substituents is NH₂    while the other is CH₃ and R₆ is H. In another embodiment, one of    the R₂ and R₃ substituents is NH₂ while the other is H and one of    the R₄ and R₅ substituents is CH₃ while the other is H, and R₆ is H.

In accordance with one embodiment of the invention a compound isprovided that could be converted by phosphorylation to an S1P receptormodulating compound. The compound has the general structure:

-   wherein R₁ is selected from the group consisting of alkyl, alkenyl,    alkynyl, alkyl (optionally substituted aryl), alkyl (optionally    substituted cycloalkyl), arylalkyl and arylalkyl (optionally    substituted aryl) R₇ is H, O, or R₁ and R₇ taken together form an    optionally substituted C₃-C₆ heteroaryl or optionally substituted    C₃-C₆ heterocyclic group; R₆ is H, C₁-C₄ alkyl or (CH₂)aryl; R₂ and    R₃ are independently selected from the group consisting of H, NH₂,    OH, C₁-C₆ alkyl, —(C₁-C₄ alkyl)OH, and —(C₁-C₄ alkyl)NH₂; R₄ and R₅    are independently selected from the group consisting of H, NH₂, OH,    C₁-C₆ alkyl, —(C₁-C₄ alkyl)OH, and C₁-C₄ alkyl)NH₂. In one    embodiment, one of the R₂ and R₃ substituents is NH₂ while the other    is CH₃ and R₆ is H. In another embodiment, one of the R₂ and R₃    substituents is NH₂ while the other is H and one of the R₄ and R₈    substituents is CH₃ while the other is H, and R₆ is H.

In accordance with one embodiment an S1P receptor modulating compound isprovided wherein the compound has the general structure:

-   wherein R₁ is selected from the group consisting of C₁-C₆ alkyl,    C₁-C₆ alkenyl, C₁-C₆ alkynyl, or C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆    alkynyl optionally substituted with OH;-   R₂ is C₅-C₁₀ alkyl, C₅-C₁₀ alkoxy, (CH₂).O(CH₂)m, C₅-C₁₀ (optionally    substituted aryl), C₅-C₁₀ (optionally substituted heteroaryl),    C₅-C₁₀ (optionally substituted cycloalkyl), C₅-C₁₀ alkoxy    (optionally substituted aryl), C₅-C₁₀ alkoxy (optionally substituted    heteroaryl) and C₅-C₁₀ alkoxy (optionally substituted cycloalkyl);-   R₃ is selected from the group consisting of H, halo, C₁-C₆ alkoxy,    C₁-C₆ alkyl, (CH₂)_(y)NH₂, (CH₂)_(y)cyano and C₁-C₆ alkylthio;-   R₄ is selected from the group consisting of hydroxy, phosphate,    methylene phosphonate, α-substituted methylene phosphonate,    thiophosphate, thiophosphonate and other phosphate anlogs and    phosphonate analogs or a pharmaceutically acceptable salt thereof;-   R₅ is selected from the group consisting of H, halo, C₁-C₄ alkyl and    haloalkyl;-   X is CR₈R₉;-   Y is selected from the group consisting of CR₈R₉, carbonyl, NH, 0 or    S;-   R₈ and R₉ are independently selected from the group consisting of H,    halo and hydroxy;-   n and m are integers independently ranging from 5-10, and-   y is an integer ranging from 0-10 with the proviso that X and Y are    not both methylene. In one embodiment a compound of the Formula IX    is provided wherein R₅ is selected from the group consisting of H,    F, methyl and ethyl. In another embodiment a compound of the Formula    IX is provided wherein X is selected from the group consisting of    CH₂, CHF, CF₂, and CHOH. In a further emboiment a compound of the    Formula IX is provided wherein-   R₁ is selected from the group consisting of CH₃, CH₂CH₃, CH₂OH,    CH₂CH₂OH and CH₂CH₂CH₂OH;-   R₂ is C₅-C₁₀ alkyl, C₅-C₁₀ alkoxy, (CH₂)_(n)O(CH₂)_(m), C₅-C₁₀    (optionally substituted aryl), C₅-C₁₀ (optionally substituted    heteroaryl) and C₅-C₁₀ (optionally substituted cycloalkyl);

R₃ and R₈ are H;

-   R₄ is selected from the group consisting of hydroxy, phosphate and    ethylene phosphonate;-   X is CH₂;-   Y is selected from the group consisting of carbonyl, NH, O and S;    and-   n and m are integers independently ranging from 5-10. In one    embodiment a compound of Formula IX is provided wherein R₁ is —CH₃,    or —CH₂CH₃; R₂ is C₅-C₁₀ alkyl; R₃ and R₅ are H; R₄ is hydroxy or    phosphate X is CH₂; and Y is selected from the group consisting of    carbonyl, NH and O.

The present invention also encompasses the pharmaceutically acceptablesalts of the compounds of the Formula IX including salts with inorganicacids, such as hydrochloride, hydrobromide and sulfate, salts withorganic acids, such as acetate, fumarate, maleate, benzoate, citrate,malate, methanesulfonate and benzenesulfonate salts, and when a carboxygroup is present, salts with metals such as sodium, potassium, calciumand aluminium, salts with amines, such as triethylamine and salts withdibasic amino acids, such as lysine. The compounds and salts of thepresent invention encompass hydrate and solvate forms.

In one embodiment, an S1P modulating compound is provided having thegeneral structure:

-   wherein R₁ is methyl or ethyl;-   R₂ is selected from the group consisting of C₅-C₁₀ alkyl,    (CH₂)_(n)O(CH₂)_(m),-   C₅-C₁₀ (optionally substituted aryl), C₅-C₁₀ (optionally substituted    heteroaryl), C₅-C₁₀ (optionally substituted cycloalkyl), C₅-C₁₀    alkoxy (optionally substituted aryl), C₅-C₁₀ alkoxy (optionally    substituted heteroaryl) and C₅-C₁₀ alkoxy (optionally substituted    cycloalkyl);-   R₄ is OPO₃H₂ or OH;-   n and m are integers independently ranging from 0 to 10;-   X is a methylene group optionally substituted with one or two    fluorine atoms or a secondary alcohol in either stereoconfiguration;-   Y is a carbonyl group, —O—, —NH— or a methylene group that is    optionally substituted with one or two fluorine atoms, or a    secondary alcohol in either stereoconfiguration, with the proviso    that X and Y are not both methylene. In one embodiment the compound    of Formula X is provided wherein R₁ is methyl or ethyl; R₂ is C₅-C₁₀    alkyl or (CH₂)_(n)O(CH₂)_(m); R₄ is OPO₃H₂ or OH; X is methylene; Y    is a carbonyl group, —O— or —NH—; and n and m are integers    independently ranging from 0 to 10. More particularly, in one    embodiment compounds of Formula X are provided wherein R₁ is methyl;    R₂ is C₅-C₈ alkyl and located in the para position; R₄ is OPO₃H₂ or    OH; X is methylene; and Y is a carbonyl group or —NH—.

In accordance with one embodiment, compounds suitable for use inaccordance with the present invention include:

-   wherein R₁ is selected from the group consisting of —CH₃, —CH₂CH₃,    CH₂OH, CH₂CH₂OH; R₃ is selected from the group consisting of H,    C₁-C₆ alkoxy and C₁-C₆ alkyl; Y is selected from the group    consisting of CHOH, CF₂, CFH, carbonyl, NH, O and S; and R₁₂ is H,    C₁-C₆ alkoxy or C₁-C₆ alkyl. More particularly, suitable compounds    include the following compounds:

The present invention also encompasses compounds general structure:

-   wherein R₁ and R₁₁ are independently selected from the group    consistng of C₁₋C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl;-   R₁₉ is selected from the group consisting of C₁-C₆ alkyl and (C₁-C₆    alkyl)OH; Q is selected from the group consisting of-   R₂ is C₅-C₁₂ alkyl, C₂-C₁₂ alkenyl (CH₂)_(n)O(CH₂)_(m), C₅-C₁₀    (optionally substituted aryl),-   C₅-C₁₀ (optionally substituted heteroaryl) and C₅-C₁₀ (optionally    substituted cycloalkyl);-   R₃ is selected from the group consisting of H, halo, C₁-C₆ alkoxy,    C₁-C₆ alkyl, (CH₂)_(n)NH₂, (CH₂)_(n)cyano and C₁-C₆ alkylthio;-   R₄ is selected from the group consisting of hydroxy,

R₅ is selected from the group consisting of H, F, methyl or ethyl;

X is CH₂, CHF, CF₂ or CHOH;

Y is selected from the group consisting of CHF, CF₂, CHOH, carbonyl, NH,O or S;

-   n and m are integers independently ranging from 0-10, with the    proviso that X and Y are not both methylene. In one embodiment R₁ is    methyl or ethyl, R₂ is C₅-C₁₀ alkyl, C₅-C₁₀ aryl or C₅-C₁₀ alkoxy,    R₃ is H, C₁-C₆ alkoxy or C₁-C₆ alkyl, R₄ is as defined immediately    above, R₅ is H, X is methylene and Y is a carbonyl group, —O— or    —NH—; or a pharmaceutically acceptable salt or tautomer thereof. In    another mbodiment Q is selected from the group consisting of-   R₂ and R₁₁ are independently selected from the group consisting of    C₅-C₁₂ akyl and C₂-C₁₂ alkenyl and R₁₅ is OH.

The compounds of the present invention are anticipated to be highaffinity agonists (or antagonists) at various sphingosine I-phosphatereceptors of the ‘Edg’ family. The compounds of the present inventionare also expected to evoke lymphopenia when introduced into rodents orhumans. Thus the compounds of the invention are immune modulators andare useful in treatment or prophylaxis of pathologies mediated bylymphocyte actions including acute or chronic rejection of tissue graftssuch as organ traslants or graft vs. host disease as well as autoimmunediseases. Autoimmunue diseases that could be treated with compounds ofthe invention include, but are not limited to: systemic lupuserythematosus, multiple sclerosis, rheumatoid arthritis, inflammatorybowel diseases including Crohn's disease and ulcerative colitis, type Idiabetes, uveitis, psoriasis and myasthenia gravis. The compounds of theinvention are useful also in treating inflammatory disorders such asatopic asthma, inflammatory glomerular injury and ischemia-reperfusioninjury.

Compounds of formula XII wherein R₁₅ is hydroxy are primary alcohols. Itis hypothesized that the hydroxy moiety of the primary alcohols isconverted to phosphates in vivo. Therefore the primary alcohol S1Panalogs of the present invention are expected to serve as prodrug formsof active S1P receptor modulating compounds. Therefore, in accordancewith one embodiment pharmaceutical compositions comprising the primaryalcohol S1P analogs of the present invention are administered to treatpatients for a variety of ailments or conditions, including the use ofthe compounds for immuno-modulation to prevent or diminish tissue graftrejection.

S1P is metabolized by a variety of conceivable routes includingphosphatases, esterases or transported into cells. The S1P signal atreceptors might be prolonged if the routes of degradation could beevaded or inhibited by S1P structural analogs. The S1P analogs of thepresent invention canbe used, in accordance with one embodiment, toinhibit or evade endogenous S1P metabolic pathways includingphosphotases, esterases, transporters and S1P acyl transferases. Forexample those S1P analogs that lack an ester bond would be resistant todegradation by endogenous esterases. One embodiment of the presentinvention is directed to compounds that function as a S1P receptoragonists and antagonists that are resistant to hydrolysis by lipidphosphate phosphatases (LPPs) or are sub-type selective inhibitors ofLPPs, and in particular are resistant to hydrolysis by sphingosine1-phosphate phosphohydrolase. Previously described S1P mimetics containa phosphate group, and thus are likely susceptible to hydrolysis byLPPs.

Alpha hydroxy phosphonates are well known phosphate mirnetics. Forexample, the compounds used clinically to treat osteoporosis(pamidronate, alendronate) are alpha hydroxy bisphosphonates that areanalogs of pyrophosphate. S1P analogs can be prepared wherein thephosphate moiety is replaced by an alpha hydroxy phosphonate.Accordingly, one aspect of the present invention is directed to lipidphosphate phosphatase resistant S1P analogs having the generalstructures of Formula IX or I wherein R₄ or R₁₅, respectively, areselected from the group consisting of

The compounds of the present invention can be used for immunomodulationas well as in antiangiogenesis therapy, most particularly as applied intherapy of neoplastic disease. In another embodiment the SP1 analogs ofthe present invention are used in the protection of female gonads duringradiation therapy such as applied to the abdomen in the course oftreatment of neoplastic diseases.

Lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidicacid (LPA), stimulate cellular proliferation and affect numerouscellular functions by signaling through G protein-coupled endothelialdifferentiation gene-encoded (S1P) receptors. Accordingly, the S1Preceptor agonists disclosed in the present invention are anticipated tohave utility in a variety of clinical settings including but not limitedto the acceleration of wound healing (including corneal wounds), thepromotion of myelination (oligodendrocyte cell function) and forimmuno-modulation. In particular, LPA has been reported (Balazs et al.Am J Physiol Regul Integr Comp Physiol, 2001 280(2):R₄₆₆-472) as havingactivity in accelerating wound closing and increasing neoepithelialthickness.

In accordance with one embodiment of the present invention apharmaceutical composition comprising one or more of the S1P receptoragonists of the present invention is administered to a mammalian species(including humans) to enhance wound repair, improve neuronal function orenhance an immune response of that species. It has also been reportedthat S1P inhibits fibrosis in various organs. Accordingly, the S1Preceptor agonists of the present invention can be used to prevent/treatdiseases associated with fibrosis of organs such as pulmonary fibrosis,interstitial pneumonia, chronic hepatitis, hepatic cirrhosis, chronicrenal insufficiency or kidney glomerular sclerosis. In one embodiment acomposition comprising an S1P receptor agonist of the present inventionis used to treat wounds, including bums, cuts, lacerations, surgicalincisions, bed sores, and slow-healing ulcers such as those seen indiabetics. Typically the composition is administered locally as atopical formulation, however other standard routes of administration arealso acceptable.

In addition it is believed that the S1P analogs of the present inventionmobilize lymphocytes and increase their homing to secondary lymphoidtissues. Thus the present analogs can be used to direct lymphocytes awayfrom transplanted organs (allografts) or healthy cells (e.g. pancreaticislets (type I diabetes), myelin sheathing (multiple sclerosis)), orother tissues that may be subjected to an undesirable immuno responseand thus decrease damage to such tissues from the immune system.

In another embodiment, the S1P receptor modulating compounds of thepresent invention are administered to a subject to treat or prevent adisorder of abnormal cell growth and differentiation as well asinflammatory diseases. These disorders include, but are not limited to,Alzheimer's disease, aberrant corpus luteum formation, osteoarthritis,osteoporosis, anovulation, Parkinson's disease, multiple sclerosis,rheumatoid arthritis and cancer. In accordance with one embodiment anS1P antagonist is administered to a patient to treat a diseaseassociated with abnormal growth. In one embodiment a compositioncomprising a compound of the general structure:

-   wherein R₁₁ is C₅-C₁₈ alkyl or C₅-C₁₈ alkenyl located in the meta or    para position;-   Q is selected from the group consisting of C₃-C₆ optionally    substituted cycloalkyl, C₃-C₆ optionally substituted heterocyclic,    C₃-C₆ optionally substituted aryl C₃-C₆ optionally substituted    heteroaryl, CH₂CH₂ and —NH(CO)—;-   R₂ is selected from the group consisting of H, C₁-C₄ alkyl and    (C₁-C₄ alkyl)OH;-   R₂₃ is selected from the group consisting of H, F, CO₂H, OH, C₁-C₆    alyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;-   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or R₂₃    together with R₂₄ and the carbon to which they are attached form a    carbonyl group; and-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and-   wherein X and R₁₂ is selected from the group consisting of O and S;    or a pharmaceutically acceptable salt or tautomer thereof and-   a pharmaceutically acceptable carrier is administered to treat a    patient suffering from a disease associated with abnormal cell    growth.

In one embodiment the compound of Formula XI is administered to treat apatient suffering from a disease associated with abnormal cell growthwherein Q is selected from the group consisting of —NH(CO)—,

-   R₂₄ is H;-   R₂₃ is H or C₁-C₄ alkyl;-   R₁₅ is selected from the group consisting of hydroxy and-   wherein R₁₂ is O or S, and in a futher embodiment Q is selected from    the group consisting of-   R₁₅ is OH;    or a pharmaceutically acceptable salt or tautomer thereof.

In addition it is believed that the S1P analogs of the present inventionmobilize lymphocytes and increase their homing to secondary lymphoidtissues. Thus the present analogs can be used to direct lymphocytes awayfrom transplanted organs (allografts) or healthy cells (e.g. pancreaticislets (type I diabetes), myelin sheathing (multiple sclerosis)), orother tissues that may be subjected to an undesirable immuno responseand thus decrease damage to such tissues from the immune system.

In accordance with one embodiment the S1P analogs of the presentinvention are used for immuno-modulation, wherein immuno-modulationrefers to an affect on the functioning of the immune system and includeslymphocyte trafficking. In accordance with one embodiment, an S1P analogof the present invention that exhibits potent agonist activity at S1P1is administered to a warm blooded vertebrate, including a human, toinduce immuno-modulation in a patient in need thereof. In one embodimentthe S1P analog is specific or has enhanced activity at the S1P1 receptorsubtype relative to one or more of the other S1P receptor subtypes.

In one embodiment of the present invention the S1P analogs of thepresent invention are used as immuno-modulators to alter immune systemactivities and prevent damage to healthy tissue that would otherwiseoccur in autoimmune diseases and in organ transplantation. Inparticular, the compounds can be administered to patients as part of thetreatment associated with organ transplantation, including pancreas,pancreatic islets, kidney, heart and lung transplantations. The S1Panalogs can be administered alone or in combo with knownimmunosuppressants such as cyclosporine, tacrolimus, rapamycin,azathioprine, cyclophosphamide, methotrexate and corticosteroids such ascortisolo, cortisone, desoxymetasone, betametasone, desametasone,flunisolide, prednisolone, prednisone, amcinomide desonide,methylprednisolone, triamcinolone, and alclometasone.

Additionally the S1P analogs of the present invention can beadministered to patients suffering from an autoimmune disease to treatthat disease. Examples of diseases considered to be autoimmune in natureare: type I diabetes, systemic lupus erythematosus, multiple sclerosis,rheumatoid arthritis, inflammatory bowel disease including colitis andCrohn's disease, glomerulonepbiitis, uveitis, Hashimoto's thyroiditis,myasthenia gravis, autoimnmune hemolytic anemia, autoimmunethrombocytopenic purpura, autoimmune hepatitis and Wegner's granuloma.

In accordance with one embodiment an immuno-modulation therapy isprovided for treating mammals, including humans, in need thereof. Themethod comprises the steps of administering to said mammal an effectiveamount of a compound represented by the formula:

-   wherein-   W is CR₂₇R₂₈ or (CH₂),NH (CO);    -   wherein R₂₇ and R₂₈ are independently selected from the group        consisting of H, halo and hydroxy;-   Y is selected from the group consisting of a bond, CR₉R₁₀, carbonyl,    NH, O or S;

wherein R₉ and R₁₀ are independently selected from the group consistingof H, halo, hydroxy and amino;

-   Z is CH₂, aryl, halo substituted aryl or heteroaryl;-   R₁₁ and R₁₆ are independently selected from the group consisting of    C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, C₂-C₁₈ alkynyl, C₅-C₁₅ alkoxy,    (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀ (heteroaryl)R₂₀,    C₅-C₁₀ (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀    alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀;

wherein R₂₀ is H or C₁-C₁₀ alkyl;

-   R₂₉ is H or halo;-   R₁₇ is selected from the group consisting of H, halo, NH₂, C₁-C₆    alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino, C₁-C₆ alkylcyano and C₁-C₆    alkylthio;-   R₂ and R₂₁ are both NH₂;-   R₃ is selected from the group consisting of H, C₁-C₆ alkyl, (C₁-C₄    alkyl)OH, and (C₁-C₄ alkyl)NH₂;-   R₂₂ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄    alkyl)OH and (C₁-C₄ alkyl)NH₂;-   R₂₃ is selected from the group consisting of H, F, CO₂H, OH, C₁-C₆    alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂;-   R₂₄ is selected from the group consisting of H, F and PO₃H₂, or R₂₃    together with R₂₄ and the carbon to which they are attached form a    carbonyl group;-   R₂₅, R₇ and R₈ are independently selected from the group consisting    of O, S, CHR₂₆, CHR₂₆, NR₂₆, and N;

wherein R₂₆ is H, F or C₁-C₄ alkyl;

-   R₁₅ is selected from the group consisting of hydroxy, phosphonate,    and

wherein R₁₂ is selected from the group consisting of O, NH and S;

X is selected from the group consisting of O, NH and S;

-   y and m are integers independently ranging from 0 to 4;-   p and q are integers independently ranging from 1 to 10;-   n is an integer ranging from 0 to 10;    or a pharmaceutically acceptable salt or tautomer thereof, with the    proviso that W and Y are not both methylene. In one embodiment the    compound has the general structure of Formula II-VII as described    herein to treat a patient by suppressing the immune system and    diminishing damage to healthy tissue that would otherwise occur in    autoimmune diseases and in organ transplantation.

In one embodiment the immuno-modulating compound has the generalstructure:

wherein R₆ is selected from the group consisting of C₁-C₁₀ alkyl and R₂and R₃ are independently selected from the group consisting of H, andNH₂ with the proviso that R₂ and R₃ are not the same, and either R₂ orR₃ is NH₂; R₂₁ is selected from the group consisting of C₁-C₆ alkyl,(C₁₄ alkyl)OH and (C₁-C₄ alkyl)NH₂; and R₁₅ is selected from the groupconsisting of hydroxy, phosphonate, and

-   wherein R₁₂ is selected from the group consisting of O, NH and S; as    well as pharmaceutically acceptable salts or tautomers of such    compounds.

The dosage to be used is, of course, dependent on the specific disorderto be treated, as well as additional factors including the age, weight,general state of health, severity of the symptoms, frequency of thetreatment and whether additional pharmaceuticals accompany the treatmentThe dosages are in general administered several times per day andpreferably one to three times per day. The amounts of the individualactive compounds are easily determined by routine procedures known tothose of ordinary skill in the art.

S1P also acts as a survival factor in many cell types. In particular S1Preceptor agonists are anticipated to have activity in protecting cellsand tissues from hypoxic conditions. In accordance with one embodimentthe S1P antagonists of the present invention are administered to treatcells and tissues exposed to hypoxic conditions, including injurysustained as a result of ischemia. In accordance with one embodiment theS1P analogs exhibiting S1P receptor antagonist activity can be used totreat ischemia reperfusion type injury. Interference with the supply ofoxygenated blood to tissuesis defined as ischemia. The effects ofischemia are known to be progressive, such that over time cellularvitality continues to deteriorate and tissues become necrotic. Totalpersistent ischemia, with limited oxygen perfusion of tissues, resultsin cell death and eventually in coagulation-induced necrosis despitereperfusion with arterial blood. A substantial body of evidenceindicates that a significant proportion of the injury associated withischemia is a consequence of the events associated with reperfusion ofischemic tissues, hence the term reperfusion injury.

The present invention is also directed to pharmaceutical compositionscomprising the S1P receptor modulating compounds of the presentinvention. More particularly, such S1P receptor agonists and antagonistscan be formulated as pharmaceutical compositions using standardpharmaceutically acceptable carriers, fillers, solublizing agents andstabilizers known to those skilled in the art Pharmaceuticalcompositions comprising the S1P receptor agonists and/or antagonists areadministered to an individual in need thereof by any number of routesincluding, but not limited to, topical, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means. The oralroute is typically employed for most conditions requiring the compoundsof the invention. Preference is given to intravenous injection orinfusion for the acute treatments. For maintenance regimens the oral orparenteral, e.g. intramuscular or subcutaneous, route is preferred. Inaccordance with one embodiment a composition is provided that comprisesan S1P analog of the present invention and albumin, more particularly,the composition comprises an S1P analog of the present invention, apharmaceutically acceptable carrier and 0.1-1.0% albumin. Albuminfunctions as a buffer and improves the solubility of the compounds.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. In accordance with oneembodiment a kit is provided for treating a patient in need ofimmuno-modulation. In this embodiment the kit comprises one or more ofthe S1P analogs of the present invention and may also include one ormore known immuno-supressants. These pharmaceuticals can be packaged ina variety of containers, e.g., vials, tubes, microtiter well plates,bottles, and the like. Other reagents can be included in separatecontainers and provided with the kit; e.g., positive control samples,negative control samples, buffers, cell culture media, etc. Preferably,the kits will also include instructions for use.

The present invention is also directed to methods for discoveringagonists and antagonists ofthe interaction between S1P and the S1Preceptor. Such compounds are identified by using an assay for detectingS1P receptor activity (such as the [γ-35 S]GTP binding assay) andassaying for activity in the presence of S1P and the test compound. Moreparticularly, in the method described by Traynor and Nahorski, 1995,Mol. Pharmacol. 47: 848-854, incorporated herein by reference, G-proteincoupling to membranes can be evaluated by measuring the binding oflabeled GTP.

For example, samples comprising membranes isolated from cells expressingan S1P polypeptide can be incubated in a buffer promoting binding of thepolypeptide to ligand (i.e. S1P), in the presence of radiolabeled GTPand unlabeled GDP (e.g., in 20 mM HEPES, pH 7.4, 100 mM NaCl, and 10 mMMgCl₂, 80 pM ³⁵S-GTP_(γ)S and 3 μM GDP), with and without a candidatemodulator. The assay mixture is incubated for a suitable period of timeto permit binding to andactivation of the receptor (e.g., 60 minutes at30° C.), after which time unbound labeled GTP is removed (e.g., byfiltration onto GF/B filters). Bound, labeled GTP can be measured byliquid scintillation counting. A decrease of 10% or more in labeled GTPbinding as measured by scintillation counting in a sample containing acandidate modulator, relative to a sample without the modulator,indicates that the candidate modulator is an inhibitor of S1P receptoractivity.

A similar GTP-binding assay can be performed without the presence of theligand (i.e. S1P) to identify agents that act as agonists. In this case,ligand-stimulated GTP binding is used as a standard. An agent isconsidered an agonist if it induces at least 50% of the level of GTPbinding induced by S1P when the agent is present at 10 uM or less, andpreferably will induce a level which is the same as or higher than thatinduced byligand.

GTPase activity can be measured by incubating cell membrane extractscontaining an S1P receptor with γ³²P-GTP. Active GTPase will release thelabel as inorganic phosphate, which can be detected by separation offree inorganic phosphate in a 5% suspension of activated charcoal in 20mM H₃PO₄, followed by scintillation counting. Controls would includeassays using membrane extracts isolated from cells not expressing an S1Preceptor (e.g., mock-transfected cells), in order to exclude possiblenon-specific effects of the candidate modulator. In order to assay forthe effect of a candidate modulator on S1P-regulated GTPase activity,cell membrane samples can be incubated with a ligand (e.g.,. S1P), withand without the modulator, and a GTPase assay can be performed asdescribed above. A change (increase or decrease) of 10% or more in thelevel of GTP binding or GTPase activity relative to samples withoutmodulator is indicative of S1P modulation by a candidate modulator.

Identified S1P receptor agonists and antagonists can be used to treat avariety of human diseases and disorders, including, but not limited tothe treatment of infections such as bacterial, fungal, protozoan andviral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson'sdisease; acute heart failure; hypotension; hypertension; urinaryretention; osteoporosis; angina pectoris; myocardial infarction; stroke;ulcers; asthma; allergy; benign prostatic hypertrophy; migraine;vomiting; psychotic and neurological disorders, including anxiety,schizophrenia, manic depression, depression, delirium, dementia, andsevere mental retardation.

EXAMPLE 1

Chemical Syntheses of S1P Analogs

To develop good mimetics for S1P, a synthetic route was designed thathad several aspects in mind (Scheme 1). First, butoxycarbonyl protectedL-serine was chosen as starting material primarily because itretrosynthetically resembled the linker region of S1P. In addition, thestarting material is a cheap and commercially available protected aminoacid. Secondly, cbemodivergence was taken into consideration. Couplingof the long chain was performed late in the synthesis so that severalchain lengths could be prepared from a common intermediate. Anotherimportant issue to address was the overwhelming insolubility of thefinal compounds. Due to this insolubility, the target molecules couldnot be purified by chromatography or crystallization methods, nor couldthey tolerate a simple workup. It was therefore necessary to design afinal step that quantitatively generated only the target product, andallowed for removal of excess reagents under vacuum. This wasaccomplished by employing trifluoroacetic acid deprotection at the endof the route.

The syntheses of the S1P analogs described in the synthetic schemes ofExample 1 were accomplished using solvents purified by filtrationthrough alumina (activity 1) and unless otherwise indicated allreactions were conducted at room teprature. All reactions were performedunder an inert atmosphere and all products were purified using 230-400mesh silica gel. Each product was analyzed by thin layer chromatography(single spot) and spectroscopic methods including ¹H NMR, ¹³C NMR, andmass spectrometry. The assigned structures of the S1P analogs wereconsistent with all spectral data obtained. All final products wereobtained as the TFA salts.

Synthesis of (2S) S1P Analogs VPC22041, 51, 53, and 63

% Yields Compound R A B C D E VPC22041 n-C₁₂H₂₅NH 100 100 91 33 100VPC22051 n-C₁₄H₂₉NH 100 100 91 41 96 VPC22053 n-C₁₄H₂₉O 100 100 91 15100 VPC22063 n-C₁₆H₃₃NH 100 100 91 26 100

Benzyl Protection of N-Boc Serine.

To a stirring solution of N-Boc-(L)-Serine (4.87 mmol) in DMF (100 mL)was added cesium carbonate (5.11 mmol) and stirring was continued 30min. Benzyl bromide (5.84 mmol) was then added and the resultingsolution was stirred 12 h. The reaction mixture was then diluted with 5ethyl acetate (25 mL), washed with lithium bromide (3×15 mL), sodiumbicarbonate (2×15 mL), and brine (2×15mL). The organic layer was driedover sodium sulfate. The solvent was then removed under reduced pressureand the resulting tan oil was purified by flash chromatography, using1:1 petroleum etherldiethyl ether, to afford the product (100%) as awhite solid. R_(f)=0.26 (1:1 petroleum ether/diethyl ether).

Phosphorylation of Resulting Alcohol.

For phosphorylation, reaction is performed in the absence of light, workup and columns are completed with as little light as possible. To asolution of the benzyl protected serine (1.98 rnuol) in 1:1 CH₂Cl₂/THF(50 mL) was added tetrazole (3.96 mmol) and the resulting mixture wasstirred 30 min. Di-tert-butyl-di-isopropylphosphoramidite (3.96 mmol)was then added and the resulting reaction mixture was stirred 15h.Hydrogen peroxide (7.92 mmol) was then added and the resulting mixturewas stirred 3 h, cooled to 0° C., and quenched by addition of aqueousNa₂S₂O₅. The resulting solution was diluted with ethyl acetate (100 mL)and extracted with 50% aqueous Na₂S₂O₅ (2×20 mL). The organic layer wasdried over sodium sulfate and the solvent was removed under reducedpressure to afford a tan oil. Flash chromatography, using 90:10CHCl₃/acetone, provided the product (97%) as a clear oil. R_(f)=0.67(90:10 CHCl₃/acetone).

Debenzylation of Phosphorylated Serine.

To a solution of the phosphorylated serine (1.55 mmol) in 200 proofethanol (25 mL) was added a catalytic amount of palladium on activatedcarbon. To the resulting solution was applied a positive pressure ofhydrogen gas and the reaction mixture was stirred 12 h. The reactionmixture was then filtered through a plug of celite eluting with methanoland the solvent was removed under reduced pressure to yield the product(91%) as a slightly yellow oil. R_(f)=0 (90:10 CHCl₃/methanol).

Coupling of Long Chain Amine with Phosphorylated Acid.

A solution of the acid (0.252 mmol), a catalytic amount of4-dimethylaminopyridine, 1-hydroxybenzotriazole hydrate (0.277 mmol),the long chain amine or alcohol (0.252 mmol), and 15 mL of CH₂Cl₂ wascooled to 0° C. with stirring. To the resulting solution at 0° C. wasadded dicyclohexylcarbodiinide (0.277 mmol) and the mixture as allowedto return to rt with stiring continuing for 12 h. The reaction mixturewas hen recooled to 0° C. and filtered. The filtrate was washed withsodium bicarbonate (3×10 mL), ammonium chloride (3×10 mL), and theorganic layers were dried over sodium sulfate. The solvent was thenremoved under reduced pressure and the resulting yellow oil was purifiedby flash chromatography to afford the product.

-   VPC22041: 33%, white solid, R_(f)=0.78 (90:10 CHCl₃/methanol).-   VPC22051: 41%, white solid, R_(f)=0.80 (90:10 CHCl₃/methanol).-   VPC22053: 15%, white solid, R_(f)=0.20 (95:5 CHCl₃/acetone).-   VPC22063: 26%, white solid, R_(f)=0.79 (90:10 CHCl₃/methanol).

Deprotection of N-Boc and phosphate groups. To a stirred solution of theprotected final product (0.072 mmol) in CH₂Cl₂ (1 mL) was addedtrifluoroacetic acid (12.98 mmol) and stirring was continued 4 h. Underreduced pressure, solvent and excess trifluoroacetic acid were removedaffording a brown oil. The oil was rinsed with ether and the solvent wasremoved under vacuum 5 times to afford the product.

-   VPC22041: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).-   VPC22051: 96%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).-   VPC22053: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).-   VPC22063: 100%, white solid, R_(f)=9 (90:10 CHCl₃/methfmol).

For S1P analog VPC22051 the PyBOP coupling procedure (as used inVPC22135) was used in place of DCC coupling. The product was obtained in15% yield as a clear oil.Synthesis of (2R) S1P Analog VPC22135

Coupling of Long Chain Amine with Protected Serine.

To a siring solution of N-Boc-(D)-Serine-OBn (0.847 mmol) in CH₂Cl₂ (20mL) was added PyBOP (0.847 mmol)followed by diisopropylethylamine (0.847mnol). After 5 min. of stirring, 1-tetradecylamine (0.847 mmol) wasadded and stirring was continued for 1 h after which time morel-tetradecylamine was added (0.254 mmol). Stirring was continued foranother 3 h and then the reaction mixture was diluted with ethyl acetate(20 mL) and washed with sodium bicarbonate (3×15 mL), ammonium chloride(2×15 mL), and the organic layer was dried over sodium sulfate. Solventswere removed under reduced pressure to afford a clear gelatinous solid,which was purified by flash chromatography, using 95:5 CHCl₃/methanol,to afford the product (68%) as a white solid. R_(f)=0.78 (95:5CHCl₃/methanol).

Benzyl Deprotection of Coupled Product.

To a solution of the coupled product (0.579 mmol) in 200 proof ethanol(15 mL) was added a catalytic amount of palladium on activated carbon.To the resulting solution was applied a positive pressure of hydrogengas and the reaction mixture was stirred 12 h. The reaction mixture wasthen filtered through a plug of celite eluting with methanol and thenthe solvent was removed under reduced pressure to yield the product(87%) as a clear oil. R_(f)=0.5 (95:5 CHCl₃/methanol).

Phosphorylation of Resulting Alcohol.

For phosphorylation, reaction is performed in the absence of light, workup and columns are completed with as little light as possible. To asolution of the alcohol (0.474 mmol) in 1:1 CH₂Cl₂/TBF (20 mL) was addedtetrazole (0.948 mmol) and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.948 mmol) was then addedand the resulting reaction mixture was stirred 15 h. Hydrogen peroxide(1.896 mmol) was then added and the resulting mixture was then stirred24 h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅. Theresulting solution was diluted with ethyl acetate (50 mL) and washedwith sodium bicarbonate (2×15 mL), water (1×15 mL), and finally brine(1×15mL). The organic layer was dried over sodium sulfate and thesolvent was removed under reduced pressure to afford a clear oil. Flashchromatography, using 90:10 CHCl₃/acetone, provided the product (100%)as a clear 20 oil. R_(f)=0.23 (90:10 CHCl₃/acetone).

Deprotection of N-Boc and Phosphate Groups.

To a stirred solution of the protected product (0.071 mmol) in CH₂Cl₂ (1mL) was added trifluoroacetic acid (12.98 mmol) and stirring wascontinued 4 h. Under reduced pressure, solvent and excesstrifluoroacetic acid were removed affording a brown oil. Rinsed oil withether and removed under vacuum 5times to afford the product (56%) as awhite solid R_(f)=0 (90:10 CHCl₃/methanol).Synthesis of (2R) S1P Analog VPC22157, 173,199, and 211

Coupling of long chain aniline with protected serine. To a stirringsolution of N-Boc-(D))-Serine-OBn (0.339 mmol) in CH₂Cl₂ (10 mL) wasadded PyBOP (0.339 mmol) followed by diisopropylethylamine (0.339 mmol).After 5 min. of stiring, the aniline (0.339 mmol) was added and stirringwas continued for 4 h. The reaction mixture was then diluted with ethylacetate (10 mL) and washed with sodium bicarbonate (3×10 mL), ammoniumchloride (2×10 mL), and the organic layer was dried over sodium sulfate.Solvents were removed under reduced pressure to afford a cleargelatinous solid, which was purified by flash chromatography to affordthe product

-   VPC22157: 77%, white solid, R_(f)=0.80 (90:10 CHCl₃/acetone).-   VPC22173: 73%, white solid, R_(f)=0.78 (90:10 CHCl₃/acetone).-   VPC22199: 65%, white solid, R_(f)=0.79 (90:10 CHCl₃/acetone).-   VPC22211: 71%, white solid, R_(f)=0.80 (90:10 CHCl₃/acetone).

Benzyl deprotection of coupled product. To a solution of the coupledproduct (0.260 mmol) in 200 proof ethanol (10 mL) was added a catalyticamount of palladium on activated carbon. To the resulting solution wasapplied a positive pressure of hydrogen gas and the reaction mixture wasstirred 12 h. The reaction rnixture was then filtered through a plug ofcelite eluting with methanol and then the solvent was removed underreduced pressure to yield the product.

-   VPC22157: 85%, clear oil, R_(f)=0.50 (95:5 CHCl₃/methanol).-   VPC22173: 60%, clear oil, R_(f)=0.55 (95:5 CHCl₃/methanol).-   VPC22199: 70%, clear oil, R_(f)=0.48 (95:5 CHCl₃/methanol).-   VPC22211: 9%, clear oil, R_(f)=0.53 (95:5 CHCl₃/methanol).

Phosphorylation of Resulting Alcohol.

For phosphorylation, reaction is performed in the absence of light, workup and columns are completed with as little light as possible. To asolution of the alcohol (0.220 mmol) in 1:1 CH₂Cl₂/THF (10 mL) was addedtetrazole (0.400 mmol) and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphornmidite (0.400 mmol) was then addedand the resulting reaction mixture was stilred 15 h. Hydrogen peroxide(0.800 mmol) was then added and the resulting mixture was then stirred24 h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅. Theresulting solution was diluted with ethyl acetate (25 mL) and washedwith sodium bicarbonate (2×10 mL), water (1×10 mL), and finally brine(1×10 mL). The organic layer was dried over sodium sulfate and thesolvent was removed under reduced pressure to afford a clear oil. Flashchromatography provided the product as a clear oil.

-   VPC22157: 84%, clear oil, R_(f)=0.23 (90:10 CHCl₃/acetone).-   VPC22173: 96%, clear oil, R_(f)=0.30 (90:10 CHCl₃/acetone).-   VPC22199: 87%, clear oil, R_(f)=0.72 (80:20 CHCl₃/acetone).-   VPC22211: 90%, clear oil, R_(f)=0.58 (80:20 CHCl₃/acetone).

Deprotection of N-Boc and Phosphate Groups.

To a stirred solution of the protected product (0.162 mmol) in CH₂Cl₂ (2mL) was added tritluoroacetic acid (25.96 mmol) and stirring wascontinued 4 h. Under reduced pressure, solvent and excesstrifluoroacetic acid were removed affording a brown oil. Rinsed oil withether and removed under vacuum 5 times to afford the product.

-   VPC22157: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).-   VPC22173: 58%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).-   VPC22199: 75%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).-   VPC22211: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).    Synthesis of (2S) S1P Analogs VPC22179 and 181

Benzyl Protection of N-Boc Serine.

To a stirring solution of N-Boc-(L)-Serine (2.44 mmol) in DMF (50 mL)was added cesium carbonate (2.56 mmol) and stirring was continued 30min. Benzyl bromide (2.92 mmol) was then added and the resultingsolution was stirred 12 h. The reaction mixture was then diluted withethyl acetate (15 mL), washed with lithium bromide (3×10 mL), sodiumbicarbonate (2×10 mL), and brine (2×10 mL). The organic layer was driedover sodium sulfate. The solvent was then removed under reduced pressureand the resulting tan oil was purified by flash chromatography, using1:1 petroleum ether/diethyl ether, to afford the product (100%) as awhite solid. R_(f)=0.26 (1:1 petroleum ether/diethyl ether).

Phosphorylation of Resulting Alcohol.

For phosphorylation, reaction is performed in the absence of light, workup and columns are completed with as little light as possible. To asolution of the benzyl protected serine (2.22 mmol) in 1:1 CH₂Cl₂/THF(100 mL) was added tetrazole (4.43 mmol) and the resulting mixture wasstirred 30 min. Di-tert-butyl-di-isopropylphosphoramidite (4.43 mmol)was then added and the resulting reaction mixture was stirred 15 h.Hydrogen peroxide (8.86 mmol) was then added and the resulting mixturewas stirred 3 h, cooled to 0° C., and quenched by addition of aqueousNa₂S₂O₅. The resulting solution was diluted with ethyl acetate (100 mL)and extracted with 50% aqueous Na₂S₂O₅ (2×20 mL). The organic layer wasdried over sodium sulfate and the solvent was removed under reducedpressure to afford a tan oil. Flash chromatography, using 90:10CHCl₃/acetone, provided the product (97%) as a cleat oil. R_(f)=0.67(90:10 CHCl₃/acetone).

Debenzylation of Phosphorylated Serine.

To a solution of the phosphorylated serine (1.55 mmol) in 200 proofethanol (25 mL) was added a catalytic amount of palladium on activatedcarbon. To the resulting solution was applied a positive pressure ofhydrogen gas and the reaction mixture was stirred 12 h. The reactionmixture was then filtered through a plug of celite eluting with methanoland the solvent was removed under reduced pressure to yield the product(91%) as a slightly yellow oil. R_(f)=0 (90:10 CHCl₃/methanol).

Coupling of Long Chain Aniline with Phosphorylated Acid.

To a stiring solution of the phosphorylated acid (0.252 mmol) in CH₂Cl₂(10 mL) was added PyBOP (0.252 mmol) followed by diisopropylethylamine(0.252 mmol). After 5 min. of strring, the aniline (0.252 mmol) wasadded and stirring was continued for 4 h. The reaction mixture was thendiluted with ethyl acetate (10 mL) and washed with sodium bicarbonate(3×10 mL), ammonium chloride (2×10 mL), and the organic layer was driedover sodium sulfate. Solvents were removed under reduced pressure toafford the product.

-   VPC22179: 43%, white solid, R_(f)=0.40 (90:10 CHCl₃/acetone).-   VPC22181: 60%, white solid, R_(f)=0.35 (90:10 CHCl₃/acetone).

Deprotection of N-Boc and Phosphate Groups.

To a stirred solution of the protected final product (0.117 mmol) inCH₂Cl₂ (1.5 mL) was added trifluoroacetic acid (19.48 mmol) and stirringwas continued 4 h. Under reduced pressure, solvent and excesstrifluoroacetic acid were removed affording a brown oil. The oil wasrinsed with ether and the solvent was removed under vacuum 5 times toafford the product.

-   VPC22179: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).-   VPC22181: 100%, white solid, R_(f)=0 (90:10 CHCl₃/methanol).    Synthesis of (2R) S1P Analog VPC22277

Tosyl Protection of the Long Chain Aniline.

To a stirring solution of the 4-decylaniline (0.428 mmol) in pyridine (3mL) under inert atmosphere at 0° C. was added tosyl chloride (0.428mmol). The reaction mixture was warmed to r.t. After 20 min., thereaction mixture was diluted with water (10 mL) and ethyl acetate (10mL). The aqueous layer was discarded and the organic layer was washedwith 1N HCl (3×10 mL), sat sodium bicarbonate (3×10 mL) and brine (2×10mL). The organic layer was dried over sodium sulfate and the solvent wasremoved under reduced pressure to yield the product (81%) as pinkcrystals, which needed no further purification. R_(f)=0.82 (90:10CHCl₃/acetone).

-   Reduction of Protected Amino Acid.

At −0° C., under inert atmosphere, N-Boc-(D)-Ser-OBz (0.678 mmol) anddiisopropylethylarnine (0.678 mmol) were added to stirng THF (3 mL).Isobutylchloroformate (0.745 mmol) was then slowly added. The reactionmixture was allowed to stir for 1 h until a precipitate was observed.The reaction mixture was then filtered and the filtrate was re-cooled to−10° C. Meanwhile, sodium borohydride (1.36 mmol) was dissolved instirring water (0.5 mL) under inert atmosphere and this mixture wascooled to −10° C. The original reaction mixture was then cannulated intothe sodium borohydride mixture slowly and the newly formed reactionmixture was brought to r.t. and stirred 1 h. The reaction mixture wasthen quenched by addition of sat. ammonium chloride (5 mL), diluted withethyl acetate (15 mL) and the aqueous layer was discarded. The organiclayer was then washed with sat ammonium chloride (3×10 mL), sat sodiumbicarbonate (3×10 mL) and finally brine (1×10 mL). The organic layer wasdried over sodium sulfate and the solvent was removed under reducedpressure to yield the crude product as a white solid. The crude productwas purified by flash chromatography, using 80:20 CHCl₃/acetone, toafford the product (42%) as a white solid. R_(f)=0.48 (80:20CHCl₃/acetone).

Coupling of Aniline with Alcohol.

To a stirring solution of the aniline (0.209 mmol) in THF (3 mL) underan inert atmosphere was added triphenylphospine (0.254 mmol), thealcohol (0.105 Omnol), and finally DEAD (0.209 mmol). The reactionmixture was stirred 12 h and then concentrated to a clear oil. Petroleumether was added to the clear oil and solid triphenylphosphine oxide wasallowed to settle on the bottom of the flask. The clear petroleum etherlayer was then pipetted off and concentrated to a clear oil. The crudeproduct was then subjected to flash chromatography, using 1:1 petroleumether/ether, to afford the final product (50%) as a white solid.R_(f)=0.83 (1:1 petroleum ether/ether).

Tosyl deprotection of the coupled product Ammonia (20 mL) was condensedin a 2-neck round bottom flask equipped with a stirbar and cold fingerthat was cooled to −70° C. under an inert atmosphere. Sodium metal (4.27mmol) was then added to the reaction mixture followed by the tosylprotected amine (0.427 mmol) in THF (8 mL). The dark blue reactionmixture was stirred for 1 h at −70° C. and was then quenched withethanol until the solution was clear/white and the reaction mixture wasthen stirred at r.t. overnight. The reaction mixture was then dilutedwith ethyl acetate (20 mL) and washed with sat. amonium chloride (3×20mL), sat. sodium bicarbonate (3×20 mL), and finally brine (1×20 mL). Theorganic layer was dried over sodium sulfate and the solvent was removedunder reduced pressure to yield the crude product as a clear oil. Thecrude product was purified by flash chromatography, using 1:1 ethylacetate/hexanes, to afford the product (40%) as a white solid.R_(f)=0.42 (1:1 ethyl acetate/hexanes).

Phosphorylation of Resulting Alcohol.

For phosphorylation, reaction is performed in the absence of light, workup and columns are completed with as little light as possible. To asolution of the alcohol (0.130 mmol) in 1: 1 CH₂Cl₂/THF (5 mL) was addedtetrazole (0.130 mmol) and the resulting mixture was stirred 30 mnm.Di-tert-butyl-di-isopropylphosphoramidite (0.130 mmol) was then addedand the resulting reaction rnixure was stirred 15 h. Hydrogen peroxide(30%, 0.044 mL) was then added and the resulting mixture was thenstirred 24 h, cooled to 0° C., and quenched by addition of aqueousNa₂S₂O₅. The resulting solution was diluted with ethyl acetate (10 mL)and washed with sodium bicarbonate (2×10 mL), water (1×10 mL), andfinally brine (1×10 mL). The organic layer was dried over sodium sulfateand the solvent was removed under reduced pressure to afford a clearoil. Flash chromatography, using 1:1 ethyl acetate/hexanes, provided theproduct (12%) as a clear oil. R_(f)=0.41 (1:1 ethyl acetate/hexanes).

Deprotection of N-Boc and Phosphate Groups.

To a stirred solution of the protected final product (0.016 mmol) inCH₂Cl₂ (0.5 mL) was added trifluoroacetic acid (6.49 mmol) and stirringwas continued 4 h. Under reduced pressure, solvent and excesstrifluoroacetic acid were removed affording a brown oil. The oil wasrinsed with ether and the solvent was removed under vacuum 5 times toafford the product (100%) as a white solid. R_(f)=0 (90:10CHCl₃/methanol).

Synthesis of (2R) S1P Analog VPC23031, 19, 65, 69, 75 and 79

% Yields Compound(s) n A B C D E F G VPC23031 4 24 66 52 100  X 90 100VPC23019 6 100  85 90 95 X 56  92 VPC23065, 69 8 34 84 84 89 100 89  86VPC23075, 79 7 66 100  100  27  93 77 100

Coupling of Aryl Halide with Terminal Alkyne.

All starting materials were thoroughly flushed with nitrogen before thereaction. To a stirring solution of the aryl halide (2.01 mmol),bis(dibenzylideneacetone) palladium (0.04 mmol), triphenylphosphine(0.10 mmol), and copper iodide (0.04 mmol) in TBF (10 mL) under inertatmosphere was added the terminal alkyne (2.21 mmol) followed bydiisopropylethylamine (8.04 mmol). The reaction mixture was then stirredat r.t. for 12 h. The reaction mixture was then diluted with ethylacetate (15 mL) and washed with sodium bicarbonate (3×15 mL), ammoniumchloride (3×15mL) and finally brine (1×15 mL). The organic layer wasthen dried over sodium sulfate. Solvents were removed under reducedpressure to afford a tan oil. Flash chromatography provided the finalproduct.

-   VPC23031: 24%, yellow oil, R_(f)=0.61(90:10 hexanes/ether).-   VPC23019: 100%, yellow oil, R_(f)=0.55 (90:10 hexanes/ether).-   VPC23065, 69: 66%, yellow oil, R_(f)=0.75 (90:10 hexanes/ether).-   VPC23075, 79: 34%, yellow oil, R_(f)=0.75 (90:10 hexanes/ether).

Reduction of the Coupled Product.

To a solution of the coupled product (1.68 mmol) in 200 proof ethanol(10 mL) was added a catalytic amount of palladium on activated carbon.To the resulting solution was applied a positive pressure of hydrogengas and the reaction mixture was stirred 12 h. The reaction mixture wasthen filtered through a plug of celite eluting with methanol and thenthe solvent was removed under reduced pressure to yield the crudeproduct

-   VPC23031: 66%, yellow solid, R_(f)=0.53 (95.5 CHCl₃/acetone).-   VPC23019: 85%, yellow solid, R_(f)=0.55 (95:5 CHCl₃/acetone).-   VPC23065, 69: 84%, yellow solid, R_(f)=0.79 (95:5 CHCl₃/acetone).-   VPC23075, 79: 100%, yellow solid, R_(f)=0.80 (95:5 CHCl₃/acetone).

Coupling of Long Chain Aniline with Protected Serine.

To a stirring solution of N-Boc-(D)-Serine-OBn (0.740 mmol) in CH₂Cl₂(20 mL) was added PYBOP (0.740 mmol) followed by diisopropylethylantine(0.740 mmol). After 5 min. of stirring, the aniline (0.740 mmol) wasadded and stirring was continued for 4 hours. The reaction mixture wasthen diluted with ethyl acetate (20 mL) and washed with 1 N HCl (3×20mL), sodium bicarbonate (3×20 mL), and finally brine (1×20 mL), and theorganic layer was dried over sodium sulfate. Solvents were removed underreduced pressure to afford a clear oil, which was purified by flashchromatography to afford the product.

-   VPC23031: 52%, clear oil, R_(f)=0.35 (dichloromethane).-   VPC23019: 90%, clear oil, R_(f)=0.61 (70:30 hexanes/ethyl acetate).-   VPC23065, 69: 84%, clear oil, R_(f)=0.82 (90:10 CHCl₃/acetone).-   VPC23075, 79: 100%, clear oil, R_(f)=0.92 (90:10 CHCl₃/acetone).

Benzyl Deprotection of Coupled Product.

To a solution of the coupled product (0.667 mmol) in 200 proof ethanol(15 mL) was added a catalytic amount of palladium on activated carbon.To the resulting solution was applied a positive pressure of hydrogengas and the reaction mixture was stirred 12 h. The reaction mixture wasthen filtered through a plug of celite eluting with methanol and thenthe solvent was removed under reduced pressure to yield the product.

-   VPC23031: 100%, clear oil, R_(f)=0.27 (70:30 hexanes/ethyl acetate).-   VPC23019: 95%, clear oil, R_(f)=0.28 (70:30 hexanes/ethyl acetate).-   VPC23065, 69: 89%, clear oil, R_(f)=0.62 (1:1 hexanes/ethyl    acetate).-   VPC23075, 79: 27%, clear oil, R_(f)=0.43 (1:1 hexanes/ethyl    acetate).

Deprotection to Afford Free Alcohol.

To a stirred solution of the N-Boc protected alcohol (0.143 mmol) inCH₂Cl₂ (2 mL) was added trifluoroacetic acid (25.96 mmol) and stirringwas continued 4 h. Under reduced pressure, solvent and excesstrifluoroacetic acid were removed affording a brown oil. The oil wasrinsed with ether and the solvent was removed under vacuum 5 times toafford the product.

-   VPC23065: 100%, white solid, R_(f)=0.2 (90:10 CHCl₃/methanol).-   VPC23075: 93%, white solid, R_(f)=0.2 (90:10 CHCl₃/methanol).

Phosphorylation of N-Boc Protected Alcohol.

For phosphorylation, the reaction is performed in the absence of light,work up and columns are completed with as little light as possible. To asolution of the alcohol (0.247 mmol) in 1:1 CH₂Cl₂/THF (15 mL) was addedtefrazole (0.495 mmol) and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.495 mmol) was then addedand the resulting reaction mixture was stirred 15 h. Hydrogen peroxide(0.989 mmol) was then added and the resulting mixture was then stirred24 h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅. Theresulting solution was diluted with ethyl acetate (25 mL) and washedwith sodium bicarbonate (3×15 mL), ammonium chloride (3×15 mL), andfinally brine (1×15 mL). The organic layer was dried over sodium sulfateand the solvent was removed under reduced pressure to afford a clearoil. Flash chromatography provided the product.

-   VPC23031: 90%, clear oil, R_(f)=0.80 (80:20 ether/ethyl acetate).-   VPC23019: 56%, clear oil, R_(f)=0.82 (80:20 ether/ethyl acetate).-   VPC23069: 89%, clear oil, R_(f)=0.85 (90:10 ether/ethyl acetate).-   VPC23079: 77%, clear oil, R_(f)=0.85 (90:10 ether/ethyl acetate).

Deprotection of N-boc and Phosphate Groups.

To a stirred solution of the protected product (0.162 mmol) in CH₂Cl₂ (2mL) was added trifluoroacetic acid (25.96 mmol) and stirring wascontinued 4 h. TUnder reduced pressure, solvent and excesstrifluoroacetic acid were removed affording a brown oil. Rinsed oil withether and removed under vacuum 5 times to afford the product.

-   VPC23031: 100%, clear oil, F_(f)=0 (90:10 CHCl₃/methanol).-   VPC23019: 92%, clear oil, R_(f)=0 (90:10 CHCl₃/methanol).-   VPC23069: 86%, clear oil, R_(f)=0 (90:10 CHCl₃/methanol).-   VPC23079: 100%, clear oil, R_(f)=0 (90:10 CHCl₃/methanol).    Synthesis of (2R) S1P Analog VPC23087 and 89:

Coupling of Aryl Halide with Terminal Alkyne.

All starting materials were thoroughly flushed with nitrogen before thereaction. To a stirring solution of the aryl halide (2.01 mmol),bis(dibenzylideneacetone) palladium (0.04 mmol), triphenylphosphine(0.10 mmol), and copper iodide (0.04 mmol) (10 mL) under inertatmosphere was added the terminal alkyne (2.21 mmol) followed bydiisopropylethylamine (8.04 mmol). The reaction mixture was then stirredat r.t. for 12 h. The reaction mixture was then diluted with ethylacetate (15 mL) and washed with sodium bicarbonate (3×15 mL), aimmoniumchloride (3×15 mL) and finally brine (1×15 mL). The organic layer wasthen dried over sodium sulfate. Solvents were removed under reducedpressure to afford a tan oil. Flash chromatography, using 70:30hexanes/ethyl acetate provided the final product (44%) as a yellowsolid. R_(f)=0.79 (70:30 hexanes/ethyl acetate).

Coupling of Long Chain Aniline with Protected Serine.

To a stirring solution of N-boc-(D)-Serine-OBn (0.288 mmol) in CH₂Cl₂(10 mL) was added PyBOP (0.288 mmol) followed by diisopropylethylamine(0.288 mmol). After 5 min. of stirring, the aniline (0.288 mmol) wasadded and stirring was continued for 4 hours. The reaction mixture wasthen diluted with ethyl acetate (10 mL) and washed with 1 N HCl (3×10mL), sodium bicarbonate (3×10 mL), and finally brine (1×10 mL), and theorganic layer was dried over sodium sulfate. Solvents were removed underreduced pressure to afford a clear oil. Flash chromatography, using70:30 hexanes/ethyl acetate provided the final product (65%) as a clearoil. R_(f)=0.64 (70:30 hexanes/ethyl acetate).

Benzyl Deprotection and Reduction of Coupled Product.

To a solution of the coupled product (0.188 mmol) in 200 proof ethanol(10 mL) was added a catalytic amount of palladium on activated carbon.To the resulting solution was applied a positive pressure of hydrogengas and the reaction mixture was stirred 12 h. The reaction mixture wasthen filtered through a plug of celite eluting with methanol and thenthe solvent was removed under reduced pressure to yield the crudeproduct as a clear oil. Flash chromatography, using 1:1 hexanes/ethylacetate provided the final product (49%) as a clear oil. R_(f)=0.51 (1:1hexanes/ethyl acetate).

Deprotection to Afford Free Alcohol.

To a stirred solution of the N-Boc protected alcohol (0.025 mmol) inCH₂Cl₂ (1 mL) was added trifluoroacetic acid (12.98 mmmol) and stirringwas continued 4 h. Under reduced pressure, solvent and excesstrifluoroacetic acid were removed affording a brown oil. The oil wasrinsed with ether and the solvent was removed under vacuum 5 times toafford the product (100%) as a white solid. R_(f)=0.2 (90:10CHCl₃/methanol).

Phosphorylation of N-Boc Protected Alcohol.

For phosphorylation, reaction is performed in the absence of light, workup and columns are completed with as little light as possible. To asolution of the alcohol (0.092 mmol) in 1:1 CH₂Cl₂/THF (10 mL) was addedtetrazole (0.183 mmol) and the resulting mixture was stirred 30 min.Di-tert-butyl-disopropylphosphoramidite (0.1 83 mmol) was then added andthe resulting reaction mixture was stirred 15 h. Hydrogen peroxide(0.367 mmol) was then added and the resulting miure was then stirred 24h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅. Theresulting solution was diluted with ethyl acetate (15 mL) and washedwith sodium bicarbonate (3×15 mL), ammonium chloride (3×15 mL), andfinally brine (1×15 mL). The organic layer was dried over sodium sulfateand the solvent was removed under reduced pressure to afford a clearoil. Flash chromatography, using 90:10 ethyl acetate/etherprovided thefinal product (93%) as a clear oil. R_(f)=0.85 (90:10 ethylacetate/ether).

Deprotection of N-Boc and Phosphate Groups.

To a stirred solution of the protected product (0.063 mmol) in CH₂Cl₂ (2mL) was added trifluoroacetic acid (25.96 mmol) and stihing wascontinued 4 h. Under reduced pressure, solvent and excesstrifluoroacetic acid were removed affording a brown oil. The oil wasrinsed with ether and the solvent was removed under vacuum 5 times toafford the product (100%) as a white solid. R_(f)=0 (90:10CHCl₃/methanol).Synthesis of (2R) Benzimidazole Compound:

Acetylation of the Aniline.

To a stiring solution of acetic anhydride (10 mL) under inert atmospherewas added octyl aniline (0.738 mmol) and stirting was continued for 1 h.Sat aqueous sodium bicarbonate was then added to neutralize and aceticacid present The aqueous solution was then extracted with ethyl acetate(3×15 mL) and the combined organic extracts were dried over sodiumsulfate and concentrated to afford the final product (100%) as a yellowsolid that was used without further purification. R_(f)=0.48 (90:10CHCl₃/acetone).

Nitration of the Acetylated Aniline.

To a stiring solution of acetic acid (1.08 mL), acetic anhydride (0.73mL), and nitric acid (0.20 mL) at −15° C. under an inert atmosphere wasadded the acetylated aniline (0.91 mmol) in approx. 1 mL of acetic acidover a period of 3 h. Reaction mixture was periodically warmed to 0° C.to avoid freezing. The reaction mixture was stirred for an additionalhour and was then diluted with ethyl acetate (10 mL) and neutralizedusing 1M NaOH and sat aqueous sodium bicarbonate. The organic layer wasremoved and the aqueous portion was washed twice more with ethyl acetate(10 mL each). The organic layers were combined and dried over sodiumsulfate and then concentrated to a yellow solid. Flash chromatography,using 95:5 CHCl₃/acetone provided the final product (100%) as a yellowsolid. R_(f)=0.68 (95:5 CHCl₃acetone).

Deacetylation of the Aniline.

To a stirring solution of the nitrated, acetylated aniline (0.62 mmol)in ethanol (2.5 mL) under an inert atmosphere was added 40% KOH (0.13mL). The reaction mixture was then heated to refiux for 1 h The solutionwas then cooled in ice and brought to pH=6 using conc. HCl. This mixturewas then concentrated to an orange solid and redissolved in ether (10mL) and washed with sat aqueous sodium bicarbonate (2×10 mL) and brine(1×10 mL). The organic layer was then dried over sodium sulfate andconcentrated to afford the final product (84%) as an orange solid thatwas used without further purification.

-   R_(f)=0.82 (95:5 CHCl₃/acetone).

Reduction of the Nitro Group.

To a stirring solution of the nitrated aniline (0.248 mmol) in aceticacid (5 mL) was added a catalytic amount of zinc dust and stirring wascontinued overnight under an inert atmosphere. The reaction mixture wasthen diluted with ether and filtered through a plug of celite under andinert atmosphere using ether to elute. Care was taken not to expose theether solution to air. The solution was then concentrated to afford thefinal product (92%) as a reddish-brown oil which was used directly inthe next step without further purification. R_(f)=0.05 (95:5CHCl₃/acetone).

Coupling of the Diamine with Protected Serene.

A solution of N-boc-(D)-Serine-OBn (0.999 mmol), PyBOP (0.999 mmol),diisopropylethylamine (0.999 mmol) in CH₂Cl₂ (25 mL) was stirred 5 mnin.under an inert atmosphere and then cannulated into a flask containingthe diamine (0.999 mmol). This reaction mixture was then stirred 12 h.The reaction mixture was then diluted with ethyl acetate (30 mL) andwashed with sat aqueous sodium bicarbonate (3×3 mL), ammonium chloride(3×30 mL), and finally brine (1×30 mL), and the organic layer was driedover sodium sulfate. Solvents were removed under reduced pressure toafford a brown oil. Flash chromatography, using 90:10 CHCl₃/acetoneprovided the final product (17%) as a brown oil. R_(f)=0.52 (90:10CHCl₃/acetone).

Benzyl Deprotection of Coupled Product.

To a solution of the coupled product (0.167 mmol) in 200 proof ethanol(10 mL) and a catalytic amount of formic acid was added a catalyticamount of palladium on activated carbon. To the resulting solution wasapplied a positive pressure of hydrogen gas and the reaction mixture wasstirred 12 h. The reaction mixture was then filtered through a plug ofcelite eluting with methanol and then the solvent was removed underreduced pressure to yield the crude product as a tan oil. Prep. platethin layer chromatography, using 90:10 CHCl₃/acetone provided the finalproduct (57%) as a tan/white solid. R_(f)=0.08 (90:10 CHCl₃/acetone).

Deprotection to Afford Free Alcohol.

To a stirring solution of the N-Boc protected alcohol (0.008 mmol) inCH₂Cl₂ (0.5 mL) was added trifluoroacetic acid (0.5 mL) and stiring wascontinued 4 h Under reduced pressure, solvent and excess triluoroaceticacid were removed affording abrown oil. The oil wasrinsed with ether andthe solvent was removed under vacuum 5 times to afford the product(100%) as a tan solid. R=0.2 (90:10 CHCl₃/methanol).

Phosphorylation of N-Boc Protected Alcohol.

For phosphorylation, reaction is performed in the absence of light, workup and columns are completed with as little light as possible. To asolution of the alcohol (0.085 mmol) in 1:1 CH₂Cl₂/THF (5 mL) was addedtetrazole (0.170 mmol) and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.170 mmol) was then addedand the resulting reaction mnixture was strred 15 h. Hydrogen peroxide(0.340 mmol) was then added and the resulting mixture was then stirred 4h, cooled to 0° C., and quenched by addition of aqueous Na₂S₂O₅.Theresulting solution was diluted with ethyl acetate (10 mL) and washedwith sodium bicarbonate (3×10 mL), ammonium chloride (3×10 mL), andfinally brine (1×10 mL). The organic layer was dried over sodium sulfateand the solvent was removed under reduced pressure to afford theproduct.

Deprotection of N-Boc and Phosphate Groups.

To a stirring solution of the protected product in CH₂Cl₂ was addedtrifluoroacetic acid and stirring was continued 4 h. Under reducedpressure, solvent and excess trifluoroacetic acid were removed affordinga brown oil. The oil was rinsed with ether and the solvent was removedunder vacuum 5 times to afford the product

EXAMPLE 2

All reactions for the synthetic schemes of Example 2 were accomplishedusing solvents purified by filtration through alumina (activity I)immediately prior to use. All reactions were performed under an inertatmosphere of nitrogen unless otherwise noted. All reagents werepurchased from either Aldrich (Milwaukee, Wis.), Sigma (St. Louis,Mos.), Acros (Pittsburgh, Pa.), Advanced ChemTech (Louisville, Ky.), orNovabiochem (La Jolla, Calif.). Merck silica gel F-254 precoated,aluminum backed plates were used for thin layer chromatography (TLC)analysis. Analtech Silica Gel GF 500 or 1000 μm precoated, glass backedplates were used for preparative TLC. Silicycle Ultra Pure Silica Gel(230-400 mesh) or Fisher Scientific Silica Gel 60 Sorbent (230400 mesh)was used for column chromatography. Each product was analyzed by TLC(single spot) and spectroscopic method including ¹H NMR, ¹³C NMR, andmass spectrometry. The nuclear magnetic resonance spectra were collectedusing a General Electric QE300 spectrometer at 300 MHz and chemicalshifts are reported in ppm. The assigned structures of the S1P analogswere consistent with all spectral data obtained.Synthesis of Imidiazole Analog

Reagents and Conditions: (i) NaH, THF 0° to R.T. 45 min., thenSelectfluor 0° to R.T., overnight, 53%; (ii) SOCl₂, MeOH, R.T., 4-6 h.;(iii) Boc₂O, TEA, CH₂Cl₂, R.T., 4 h; (iv) 2,2-dinethoxypropan,p-toluenesulfonic acid, CH₂Cl₂, R.T., 2 h, 62% (3 steps) (v) LiCi,NaBH₄, EtOH/THF (3:2), 0° to R.T., 4 h, 89%; (vi) PCC, CH₂Cl₂, R.T., 6h.; (vii) DBU, LiCl, CH₃CN, R.T., overnight, 40% (2 steps); (viii) Dowex50×8, EtOH, R.T. 24 h., 80%; (ix) PCC, CH₂Cl₂, R.T. , 6 h. (x) NaClO₂,NaH₂PO₄.H₂O, t-butanol, 2-methyl-2-butene; (xi) poctyl aniline, PyBOP,DIEA, CH₂Cl₂, R.T., overnight; (xii) H₂, 10% Pd/C, EtOH, R.T. overnight;(xiii) TMSBr, CH₂Cl₂, R.T., 4 h., then 95% CH₃OH in H₂O, R.T., 1 h.

2-Bromo-1-(4octyl-phenyl)-ethanone (1).

To a flame dried round bottom flask equipped with a magnetic stirbarunder an inert atmosphere was added AlCl₃ (5.47 g, 41 mmol) followed by1,2-dichloroethane (22 mL). The stiring suspension was then brought to0° C. and 1-phenyloctane (7.99 mL, 36 mmol) was added in one portion.Bromoacetyl bromide (3.75 mL, 43 mmol) was then added dropwise over aperiod of 10 minutes. Upon completing addition of the acid bromide, thereaction mixture was brought to rt and stirred for 2 h The reactionmixture was then quenched carefully by slow addition of H₂O (36 mL)without ever letting the reaction mixture exceed 45° C. producing asuspension of solid white precipitate. The aqueous layer of the quenchedreaction mixture was discarded and the organic phase was washed oncewith 10% HCl (10 mL), washed once with H₂O (10 mL), and dried overmagnesium sulfate. The dried organic phase was then concentrated invacuo to a green/brown oil. Recrystalization from MeOH/H₂O provided theproduct 1 (6.36 g, 57%) as white needles in three crops. R_(f)=0.21(1:19 EtOAc/hexanes).

2-Amino-3-hydroxy-2-methyl-propionic acid methyl ester (2).

A strring solution of α-methyl-DL-Serine (1 g, 8.39 mmol) in MeOH (40mL) in a flame dried round bottom flask under an inert atmosphere wascooled to 0° C. and SOCl₂ (1.84 mL, 25.19 mmol) was slowly added. Afteraddition of the SOCl₂ was complete, the reaction mixture was stirred 12h at rt and then concentrated in vacuo to a white solid that was useddirectly in the next reaction.

2-tert-Butoxycarbonylamino-3-hydroxy-2-methyl-propionic acid methylester (3).

To the crude product obtained in the above reaction was slowly added sataq. NaHCO₃ (12.5 mL) followed by solid NaHCO₃(500 mg) and the reactionmixture was stirred 30 min under an inert atmosphere. TBF (12.5 mL) wasthen added to the reaction mixture followed by di-tert-butyl dicarbonate(1.83 g, 8.39 mmol) and strring at rt was continued for 12 h Thereaction mixture was then diluted with H₂O (20 mL) and extracted withEtOAc (3×20 mL). The combined EtOAc extracts were dried over sodiumsulfate and concentrated in vacuo to a thick white paste. To this pastewas added hexanes which produced 3 (630 mg, 32% for 2 steps) as a whiteprecipitate which was collected by filtration. R_(f)=0.35 (1:1EtOAclhexanes).

2,2,4-Trimethyl-oxazolidine-3,4-dicarboxylic acid 3-tert-butyl ester4-methyl ester (4).

To a stirring solution of 3 (9.342 g, 40 mmol) in acetone (115 mL) in aflame dried round bottom flask under an inert atmosphere was added2,2-dimethoxypropane (66 mL). To this solution was added BF₃-OEt₂ (0.30mL, cat) and stirring was continued at rt for 2 h. The reaction mixturewas then concentrated in vacuo to an orange oil which was purified byflash chromatography to provide 4 (9.392 g, 85%) as a white solid.R_(f)=0.55 (1:3 EtOAc/hexanes). Compound was observed as an unevenmixture of rotomers.

2,2,4-Trimethyl-oxazolidine-3,4-dicarboxylic acid 3-tert-butyl ester(5).

To a stirring solution of 4 (9.392 g, 34 mmol) in THF (65 mL) and H₂O(35 mL) under an inert atmosphere was added solid LiOH—H₂O (1.426 g, 34mmol) in one portion. The reaction mixture was heated to 90° C. andstirred 8 h at which point the reaction mixture was cooled to rt. Thecrude reaction mixture was washed with Et₂O (3×50 mL) and the Et₂Oextracts were discarded. The aqueous solution was then acidified with 2MKHSO₄ until a white precipitate began to form on addition, pH=5. Theacid was added dropwise until the precipitate persisted and the aqueoussolution was extracted with Et₂O (50 mL). After extraction, two additiondrops of acid were added to the aqueous layer and it was again extractedwith Et₂O (25 mL). The Et₂O extracts were combined and quickly backextracted with 1M NaOH (15 mL). The organic phase was then dried oversodium sulfate and concentrated in vacuo to give 5 (7.458 g, 85%) as awhite solid which was used without further purification. Compound wasobserved as an uneven mixture of rotomers.

2,2,4-Trimethyl-4-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-oxazolidine-3-carboxylicacid tert-butyl ester (6).

To a flame dried round bottom flask equipped with a magnetic stirbarunder an inert atmosphere was added 5 (3.00 g, 11.6 mmol) followed byabsolute EtOH (33 mL) and Cs₂CO₃ (1.93 g, 5.9 mmol). This mixture wasthen shaken 30 min at which time all of the suspended Cs₂CO₃ haddisappeared. The reaction mixture was then concentrated in vacuo to awhite solid at which time DMF (60 mL) was added. To the stirringsolution was added a solution of 1 (3.60 g, 11.6 mmol) in DMF (5 mL).The resulting solution was stirred 4 h and concentrated to a light brownsolid. To the light brown solid was added EtOAc (50 mL) and thesuspended CsBr was filtered off and washed with EtOAc. The filtrate wasthen concentrated to a light brown foam which was subsequently dissolvedin xylenes (195 mL) in a round bottom flask equipped with a Dean-Starktrap (filled with xylenes) and a reflux condenser. To this solution wasadded NH₄OAc (1.74 g, 22.6 mmol) and the reaction mixture was brought to105° C. and stirred 3 h at which time the reaction would progress nofurther. The crude reaction mixture was then concentrated in vacuo to ared oil. To the oil was added EtOAc (200 mL) and this solution waswashed with sat. aq. NaHCO₃ (3×50 mL) followed by brine solution (1×50mL). The organic phase was then dried over sodium sulfate andconcentrated to a red oil which was subjected to flash chromatography togive 6 (1.074 g, 20%) as a white solid. R_(f)=0.45 (6:4 Et₂O/petroleumether).

2-Amino-2-[5-(4-octyl-phenyl)-IH-imidazol-2-yll-propan-1-ol (VPC24241).

To a flame dried round bottom flask equipped with a magnetic stirbarunder an inert atmosphere was added 6 (973 mg, 2.07 mmol) followed byMeOH (20 nL) and p-TsOHH₂O (1.22 g, 6.42 mmol). This mixture was thenheated to reflux, stirred 3 h, cooled to 0° C., and quenched by slowaddition of sat. aq. NaHCO₃ (20 mL). This solution was then diluted withEtOAc (30 mL) and the aqueous layer was discarded. The organic phase waswashed with sat aq. NaHCO₃ (1×20 mL), washed with 1M NaOH (1×20 mL),dried over sodium sulfate, and concentrated to an orange oil. To thisoil was added Et₂O which produced VPC24241 (408 mg, 60%) as a whiteprecipitate which was collected by filtration.

{2-Hydroxy-1-methyl-1-15(4-octl-phenyl)IH-imidazol-2-yl]-ethyl}-carbamicacid tert-butyl ester (7).

To a vigorously stirring solution of VPC24241 (70 mg, 0.213 mmol) in TLF(4 mL) and H₂O (2 mL) was added Na₂CO₃ (198 mg, 1.87 mmol) followed bydi-tert-butyl dicarbonate (214 mg, 0.98 mmol) and the resulting solutionwas stirred 12 h at rt The reaction mixture was then diluted with EtOAc(20 mL) and washed with saturated aq. NaHCO₃ (2×15 mL). The organicphase was dried over sodium sulfate and concentrated in vacuo to a clearoil which solidified to a white solid under vacuum. This white solid wasthen subjected to flash chromatography to produce 7 (52 mg, 57%) as awhite solid. R_(f)=0.50 (1:1 EtOAc/hexanes).

{2(Di-tert-butoxy-phosphoryloxy)-1-methyl-1-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-ethyl}-carbamicacid tert-butyl ester (8).

To a solution of 7 (33 mg, 0.077 mmol) in 1:1 CH₂Cl₂/THF (3 mL) wasadded a 3% solution of tetrazole in acetonitrile (0.44 mL, 0.154 mmol)and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.05 mL, 0.154 mmol) was thenadded and the resulting reaction mixre was stirred 12 h. To thissolution was added 30% hydrogen peroxide (0.04 mL, 0.308 mmol) and theresulting mixture was stirred 3 h, cooled to 0° C., and quenched byaddition of aqueous Na₂S₂O₅. The resulting solution was diluted withethyl acetate (10 mL) and washed with saturated aq. NaHCO₃ (2×5 mL). Theorganic layer was dried over sodium sulfate and the solvent was removedunder reduced pressure to afford a clear oil. Flash chromatography,using 1 :1 EtOAc/hexanes, provided 8 (22 mg, 46%) as a clear oil.R_(f)=0.45 (1:1 EtOAc/hexanes).

{2-(D)i-tert-butoxy-thiophosphoryloxy)-1-methyl-1-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-ethyl}-carbamicacid tert-butyl ester (9).

To a solution of 7 (19 mg, 0.044 mmol) in 1:1 CH₂Cl₂/THF (2 mL) wasadded a 3% solution of tetrazole in acetonitrile (0.26 mL, 0.089 mmol)and the resulting mixture was stirred 30 min.Di-tert-butyl-di-isopropylphosphoramidite (0.03 mL, 0.089 mmol) was thenadded and the resulting reaction mixture was stirred 12 h. To thissolution was added elemental sulfur (excess) and the resulting mixturewas stirred 12 h The resulting solution was diluted with ethyl acetate(7 mL) and washed with saturated aq. NaHCO₃ (2×3 mL). The organic layerwas dried over sodium sulfate and the solvent was removed under reducedpressure to afford a clear oil with yellow tint. Flash chromatography,using 1:3 EtOAc/hexanes, provided 9 (13 mg, 46%) as a clear oil.R_(f)=0.40 (1:3 EtOAc/hexanes).

Phosphoric acidmono-{2-amino-2-[5-(4-octyl-phenyl)-1H-imidazol-2-yl]-propyl} ester(VPC24287).

To a stirring solution of 8 (22 mg, 0.035 mmol) in CH₂Cl₂ (1 mL) wasadded trifluoroacetic acid (1 mL) and stirring was continued 4 h.Solvent and excess trifluoroacetic acid were removed in vacuo to afforda brown oil. The oil was diluted with ether and concentrated in vacuo 5times on a rotary evaporator to afford a white solid which was placed ina fritted fimnel and washed with cold ether producing VPC24287 (13 mg,91%) as a powdery white solid. R_(f)=0 (4:1 CHCl₃/methanol).

Thiophosphoric acidO-{2-amino-2-[5-(4octyl-phenyl)-1H-imidazol-2-yl]-propyl} ester(VPC24289).

To a stiring solution of 9 (13 mg, 0.020 mmol) in CH₂Cl₂ (1 mL) Wasadded benzenethiol (0.042 mL, 0.40 mmol) followed by bromotrimethylsilane (0.05 mL, 0.40 mmol) and finally trirluoroacetic acid (1 mL) andstiring was continued 6 h To quench the reaction mixture, water (0.5 mL)was added and the resulting solution was stirred 30 min. Solvent andexcess reagents were removed in vacuo to afford a brown oil. The oil wasdiluted with ether and concentrated in vacuo 5 times on a rotaryevaporator to afford a light tan solid which was placed in a frittedfunnel and washed with cold ether and a small amount of cold waterproducing VPC24289 (8 mg, 94%) as a powdery white solid. R_(f)=0 (4:1CHCl₃/metha.

Synthetic Scheme for Synthesis of Additional Imidizole Compounds

Reagents and Conditions: (i) Br₂, 1:1 dioxane/ether, CH₂Cl₂, rt, 1 h,66%; (ii) 2,2-DMP, p-TsOH, DMF, rt, 12 h, TEA, rt, 10 min; (iii)(Boc)₂O, NaHCO₃, THF/H₂O, rt, 12 h, 69% (2 steps); (iv) (COCI)₂, DMSO,TEA, CH₂Cl₂, −78° C. to rt, 4 h, 74%; (v) NaClO₂, NaH₂PO₄.H₂O,2-methyl-2-butene, tBuOH/H₂O, rt, 1 h, 95%; (vi) Cs₂CO₃, EtOH, rt, 1 h;1, DMF, rt, 12 h, (vii) NH₄OAc, xylenes, 1 10° C., 12 h, 36% (2 steps);(viii) Pd(dba)₂, Ph₃P, CuI, DIEA, TBF, rt, 12 h, 68%; (ix) H₂, 10% Pd/C,EtOH, rt, 12 h, (x) 1:1 TFA/CH₂Cl₂, rt, 6 h, (xi) DOWEX 50×8, EtOH, rt12 h; (xii) tetrazole, di-tert-butyl diisopropylphosphoramidite,CH₂C₁/THF, rt 12 h; H₂₀ ₂, rt 3 h; (xiii) tetrazole, di-tert-butyldiisopropylphosphoramidite, CH₂Cl_(2/)THF, rt, 12 h; S₈, rt, 3 h; (xiv)1:1 TFA/CH₂Cl₂, rt 4 h; (xv) benzenethiol, TMSBr, 1:1 TFA/CH₂Cl₂, rt, 4hSynthetic Scheme for Synthesis of Alpha Substituted PhosphonateCompounds

Reagents and Conditions: (i) NaH, ThF 0° to R.T. 45 min., thenSelectfluor 0° to R.T., overnight, 53%; (ii) SOCl₂, MeOH, R.T., 4-6 h.;(iii) Boc₂O, TEA, CH₂Cl₂, R.T., 4 h.; (iv) 2,2-dimethoxypropane ,p-toluenesulfonic acid, CH₂Cl₂, R.T., 2 h., 62% (3 steps) (v) LiCl,NaBH₄, EtOH/THF (3:2), 0° to R.T., 4 h, 89%; (vi) PCC, CH₂Cl₂, PT., 6h.; (vii) DBU, LiCl, CH₃CN, R.T., overnight, 40% (2 steps); (viii) Dowex50×8, EtOH, R.T. 24 h., 80%; (ix) PCC, CH₂Cl₂, R.T. , 6 h. (x) NaClO₂,NaH₂PO₄.H₂O, t-butanol, 2-methyl-2-butene; (xi) p-octyl aniline, PyBOP,DIEA, CH₂Cl₂, R.T., overnight; (xii) H₂, 10% Pd/C, EtOH, R.T. overnight;(xiii) TMSBr, CH₂Cl₂, R.T., 4 h., then 95% CH₃OH in H₂O, R.T., 1 h.

[(Diethoxy-phosphoryl)-fluoro-methyl]-phosphonic acid diethyl ester(31).

To a slurry of 95% NaH (9 mg, 0.375 mmol) in TF (1.5 mL) was addedtetraethyl methylene diphosphonate, (30) (100 mg, 0.347 mmol) at 0° C.The mixture was allowed to warm to room temperature and stirred for 45minutes. The mixture was subsequently cooled to 0° C. and Selectfluor(153 mg, 0.432 mmol) was added in one portion. The mixture was allowedto warm to room temperature and stirred for 1 hr. The reaction mixturewas concentrated in vacuo and purified by column chromatography on SiO₂(3% MeOH in EtOAc) to yield 56 mg (53%) of a clear liquid.

2-Amino-3-hydroxy-propionic acid methyl ester (33).

To a solution of D-serine (5 g, 47.58 mmol) in methanol (100 mL),stirring under N₂ (g) at 0° C., was added thionyl chloride (20.8 mL,285.5 mmol) dropwise. The reaction mixture was allowed to warm to roomtemperature, stirred for 4-6 hours, then concentrated under reducedpressure. The crude material was reconstitutedin Et₂O and concentrated,in the same manner. This was repeated numerous times until SOCl₂ couldnot be detected. The crude material was confirmed by NMR experiments andcarried on to the following step.

2-tert-Butoxycarbonylamino-3-hydroxy-propionic acid methyl ester (34).

To a solution of the crude methyl ester serine (33) in CH₂Cl₂ (100 mL),stirring under N₂ (g), was added di-tert-butyl pyrocarbonate (11.420 g,52.34 mmol) and triethyl amine (16.6 mL, 118.95 mmol). The reactionmixture was allowed to stir at room temperature for 4 hours, then pouredover NH₄Cl at 0° C. The organic layer was extracted with 10% HCl (2×),then NaHCO₃ and brine. The organic layer was then dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. The crudematerial was again carried on to the following step.

2,2-Dimethyloxazolidine-3,4-dicarboxylic acid 3-tert-butyl ester4-methyl ester (35).

To a solution of (34) in CH₂Cl₂ stirring under nitrogen at 0° C., wasadded 2,2-dimethoxypropane (29,5 mL, 237.9 mmol) and p-toluene sulfonicacid monohydrate (9.050 g, 47.58 mmol). The mixture was removed from theice bath after 15 minutes and stirred at room temperature for 1.5 hours.The reaction mixture was poured into 50 mL of saturated NaHCO_(3 (aq))and extracted with diethyl ether (3×50). The organic layer was extractedwith NaHCO₃ and brine, then dried over anhydrous MgSO₄, filtered andconcentrated under reduced pressure. The crude material was purified byflash chromatography on SiO₂ (1:1 EtOAc/Hexanes) to yield 7.659 g (62%,3 steps) of a clear liquid.

4-Hydroxymethyl-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butylester (36).

To a mixture of NaBH₄ (2.247 g, 59.08 mmol) and LiCl (2.505 g, 59.08mmol) in EtOH (42 mL), siring under nitrogen at 0° C., was added (35)(7.659 g, 29.54 mmol) in THF (30 mL) dropwise. This mixture was allowedto warm to room temperature and continued stirring for 48 hours. Theprecipitate was filtered and washed with ethanol. The washings wereconcentrated and extracted with EtOAc. The organic layer was then washedwith brine and dried over anhydrous Na₂SO₄. Column chromatography onSiO₂ (1:1 EtOAc/Hexanes) was utilized to purify 6.101 g (89%) of thetitle compound as a white solid.

4-Formyl-2,2-dimethyl-oxazolldine3-carboxylic acid tert-butyl ester(37).

To a solution of (36) (80 mg, 0.346 mmol) stirring in CH₂Cl₂ (2 mL),under a nitrogen atmosphere, was added pyridinium chlorochromate (150mg, 0.694 mmol). The reaction mixture was allowed to stir overnight thenfiltered through a plug of silica gel. The crude aldehyde was carried onto the following step.

4-[2(Diethoxy-phosphoryl)-2-fluoro-vinyl]-2,2-dimethyl-oxazolidine-3-carboxylicacid tert-butyl ester (38). To a stirred suspension of LiCl (18 mg,0.416 mmol) in dry acetonitrile (3.5 mL), under nitrogen at roomtemperature, were added diphosphonate (31) (127 mg, 0.416 mmol), DBU(0.05 mL, 0.347 mmol) and garner's aldehyde (37) (80 mg, 0.347 mmol).The reaction mixture was allowed to stir overnight then concentrated invacuo. The crude material was isolated by column chromatography on SiO₂,(1:1 EtOAc/Hexanes) to yield 47 mg (40%, two steps) of a clear liquid.

(3-tert-Butoxycarbonylamino-1-fluoro4 hydroxy-but-1-enyl)-phosphonicacid diethyl ester (39).

To compound (38) (47 mg, 0.123 mmol) stirring in EtOH (1 mL) was addedDowex 50×8 (150 mg), which was washed with EtOH and dried. The reactionwas allowed to stir under nitrogen and at room temperature for 24 hours.The reaction mixture was filtered and the precipitate washed with excessEtOH, then concentrated in vacuo. The crude material was purified bycolumn chromatography on SiO₂ (1:1 EtOAc/Hexanes) to yield 34 mg of theexpected product.

EXAMPLE 3

[γ-35 S]GTP Binding Assay for Measuring S1P Activity

Transient Expression in HEK293T Cells.

Human or mouse S1P5 DNA was mixed with an equal amount of DNA encoding arat Gi2R protein as well as DNAs encoding cow β1 and γ2 proteins andused to transfect monolayers of HEK293T cells using the calciumphosphate precipitate method. After 60 h, cells were harvested, andmicrosomes were prepared, aliquoted, and stored at −70° C. until use.

[γ-35 S]GTP Binding.

Briefly, 5 ug of membranes from S1P expressing HEK293T cells wasincubated in 0.1 mL of binding buffer (in mM: HEPES 50, NaCl 100, MgCl₂5), pH 7.5, containing 5 ug of saponin, 10 uM GDP, 0.1 nM [γ-35 S]GTP(1200 Ci/mmol), and test lipid. After incubating for 30 min at 30° C.,bound radionuclide was separated from free by filtration through WhatmanGF/C paper using a Brandel Cell Harvester (Gaithersburg, Md.).

Stable Expression in RH₇₇₇₇ Cells.

Rat hepatoma RH₇₇₇₇ cell monolayers were transfected with human or mouseS1P5/pCR_(3.1) DNA using the calcium phosphate precipitate method, andclonal populations expressing the neomycin phosphotransferase gene wereselected by addition of Ge-neticin (G418) to the culture medium. TheRH₇₇₇₇ cells were grown in monolayers at 37° C. in a 5% CO₂/95% airatmosphere in growth medium consisting of 90% MEM, 10% fetal bovineserum, 2 mM glutamine, and 1 mM sodium pyruvate.

Measurement of cAMP Accumulation.

Assay of cAMP accumulation was performed as described previously (See Imet al., J. Biol. Chem. 275, 14281-14286 (2000), the disclosure of whichis incorporated herein). Assays were conducted on populations of 5×10⁵cells stimulated with 1 uM forskolin in the presence of thephosphodiesterase inhibitor isomethylbutylxanthine (IBMX, 1 mM) for 15min. cAMP was measured by automated radioimmunoassay. The GTPγS studieswere performed using zebrafish S1P1 overexpressed rat RH-7777 and humanhS1P1, hS1P2, hS1P3 and hS1P5 overexpressed human HEK293 cells. Table 1shows the EC₅₀ values for each of the S1P analogs at S1P receptors:S1P1, S1P2, S1P3 and S1P5.In addition to testing the human S1P receptors(hS1P1, hS1P2, hS1P3 and hS1P5), a zebrafish S1P receptor (zS1P1) andmouse S1P (mS1P5) were also tested. TABLE 1 EC ₅₀ Values (nM) for S1PAnalogues at Recombinant S1P Receptors zS1P₁ hS1P₁ hS1P₃ hS1P₂ hS1P₅mS1P₅ S1P 54.6 0.9 1.1 2.9 43.9 12.7 VPC22041 2053.0 598.4 845.4 973.2645.5 >5000 VPC22051 >5000 322.1 601.9 2760.0 >5000 >5000 VPC22053 >5000397.0 862.4 2685.0 1606.0 2006.0 VPC22063 >5000 1805.0 878.6 >50001220.0 1326.0 VPC22135 1625.0 12.7 50.8 2107.0 >5000 1821.0S1P increases GTPγS binding significantly (2-5-fold) at each receptorwith EC₅₀ values from 1 to 55 nM. The synthetic series consisted of fivedihydro S1P of the general formula:

wherein

-   VPC22041 (2S): R₁ is NH(CH₂)₁₁CH₃, R₂ is NH₂ and R₃ is H;-   VPC22053 (2S): R₁ is O(CH₂)₁₃CH₃, R₂ is NH₂ and R₃ is H;-   VPC22051 (2S): R₁ is NH(CH₂)₁₃CH₃, R₂ is NH₂ and R₃ is H;-   VPC22063 (2S): R₁ is NH(CH₂)₁₅CH₃, R₂ is NH₂ and R₃ is H; and-   VPC22135 (2R): R₁ is NH(CH₂)₁₃CH₃, R₂ is H and R₃ is NH₂.

The amide-containing compounds contained alkyl chains of 12 (VPC22041),14 (VPC22053), or 16 (VPC22063) carbons, and the 2′-amino group was inthe natural configuration (S), except for VPC22135, wherein the 2′-aminowas in the (R) configuration. VPC22053 and VPC22135 are an enantiomericpair, while VPC22051 is the ester-containing equivalent of VPC22053 (seeScheme 4). All of these compounds had significant agonist activity ateach of the S1P receptors, although none were as potent as S1P itself(see Table 1). In particular, on the S1P5 transfected HEK293 cells, thefive mimetics showed EC₅₀'s of approximately 1 ℏM, where as the EC₅₀ ofS1P itself on the same cells is closer to 10 m. However, one compound,VPC22135, approached the potency of S1P at both the human S1P1 and humanS1P3 receptors. Curiously, this compound has the amino group in theunnatural (R) configuration. Its enantiomer, VPC22053, was more than 1log order less potent at both the S1P1 and S1P3 receptors. The resultsobtained for the S1P1transfected RH-7777 cells showed a preference forbinding with the 18 carbon backbone mimetic compounds (identical to S1P)over the 16 and 20 carbon backbone mimetic compounds.

Assay of phenyl imidazole compounds vpc24287 (phosphate) and vpc24289(phosphothionate) at individual human sphingosine 1-phosphate (S1P)receptors was also conducted.

Methods: Human recombinant S1P receptor type DNAs were mixed with DNAsencoding human Gαi2, cow β1 and cow γ2 proteins and introduced intocultured HEK293T cells by transfection. After about 48 hours, cells wereharvested and crude membranes prepared. Ligand driven binding of anon-hydrolyzable GTP analog, GTP[γ-³⁵S], was measured in a rapidfiltration assay. Details of the assay are found in: Brinkmann, V.,Davis, M. D., Heise, C. E., Albert, R, Cottens, W., Hof, R., Bruns, C.,Prieschl, E., Baumruker, T., Hiestand, P., Foster, C. and Lynch, Y R.The immune modulator, FTY720, targets sphingosine 1-phosphate receptors.J. Biol. Chem. 277: 21453-21457 (2002). Total counts per minute weredetermined for S1P, vpc24287 and vpc24289 activation of the S1P receptorsubtypes with the maxinal counts received by S1P designated as 100%activation of the S1P receptor. The results are provided in FIG. 6A-6Ddemonstrating vpc24287 and vpc24289 activation of the S1P receptorsubtypes relative to S1P.

EXAMPLE 4

Biological Assay of the Synthesized Mimetics

An additional series of compounds was tested using the GTPCS bindingassay described in Example 2 and in 1 h et al., J. Biol. Chem. 275,14281-14286 (2000), the disclosure of which is incorporated herein). Thecompounds tested for binding at human S1P receptors (hS1P1, hS1P2,hS1P3, hS1P4 and hS1P5) have the general structure:

wherein for

-   VPC23019: R₅ is (CH₂)CH₃, R₂ is H, R₃ is NH₂ and R₄ is phosphate;-   VPC23031: R₅ is (CH₂)₇CH₃, R₂ is H, R₃ is NH₂ and R₄ is phosphate;-   VPC23065: R₅ is (CH₂)₉CH₃, R₂ is H, R₃ is NH₂ and R₄ is hydroxy;-   VPC23069: R₅ is (CH₂)₉CH₃, R₂ is H, R₃ is NH₂ and R₄ is phosphate;-   VPC23075: R₅ is (CH₂)₈CH₃, R₂ is H, R₃ is NH₂ and R is hydroxy;-   VPC23079: R₅ is (CH₂)₈CH₃, R₂ is H, R₃ is NH₂ and R₄ is phosphate;    or have the general structure:    wherein for-   VPC23087: R₅ is (CH₂)₇CH₃, R₂ is H, R₃ is NH₂ and R₄ is hydroxy;-   VPC23089: R₅ is (CH₂)₇CH₃, R₂ is H, R₃ is NH₂ and R₄ is phosphate;

Each of the compounds tested (VPC 23019, 23031,23065, 23069, 23087,23089, 23075, 23079) failed to show significant activity at the S1P2receptor. Compounds VPC23065, VPC23087 and VPC23075 are primary alcoholsand thus lack the phosphate headgroup. Yet several of these compoundsexhibit activity at S1P receptors (See FIGS. 2A, 2B, 2C, 3A, 3B, 3C and4C) and each of these compounds shows good agonist activity at the S1P4receptor.

The GTP_(C)S binding assay revealed that VPC23031, VPC23019, VPC23089are inverse agonists (antagonists) of the S1P3 receptor (See FIGS. 1Aand 4A), but this inverse agonism becomes agonism when the alkyl chainlength is 9 carbons (VPC23079) or 10 (VPC23069), see FIGS. 2A and 3A.

VPC23089 and VPC23019 are isomers, with the VPC23089 compound having thealkyl chain ortho and the VPC23019 compound meta; in both cases thealkyl chain has 8 carbons, but surprisingly, when one goes from ortho tometa, antagonism at S1P1 is realized (compare FIG. 1A with thecompetition curve FIG. 5A).

1. A compound represented by the formula:

wherein W is CR₂₇R₂₈ or (CH₂)_(n)NH (CO); wherein R₂₇ and R₂₈ areindependently selected from the group consisting of H, halo and hydroxy;Y is selected from the group consisting of a bond, CR₉R₁₀, carbonyl, NH,O or S; wherein R₉ and R₁₀ are independently selected from the groupconsisting of H, halo, hydroxy and amino; Z is CH₂, aryl, halosubstituted aryl or heteroaryl; R₁₁ and R₁₆ are independently selectedfrom the group consisting of C₅-C₁₂ alkyl, C₅-C₁₂ alkenyl, C₅-C₁₂alkynyl, C₅-C₁₂ alkoxy, (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀(heteroaryl)R₂₀, C₅-C₁₀ (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀; wherein R₂₀ is Hor C₁-C₁₀ alkyl; R₂₉ is H or halo; R₁₇ is selected from the groupconsisting of H, halo, NH₂, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino,C₁-C₆ alkylcyano and C₁-C₆ alkylthio; R₂, and R₂₁ are both NH₂; R₃ isselected from the group consisting of H, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH,nd (C₁-C₄ alkyl)NH₂; R₂₂ is selected from the group consisting of C₁-C₆alkyl, (C₁-C₄ alkyl)OH and C₁-C₄ aIkyl)NH₂; R₂₃ is selected from thegroup consisting of H, F, CO₂H, OH, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and(C₁-C₄ alkyl)NH₂; R₂₄ is selected from the group consisting of H, F andPO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they areattached form a carbonyl group; R₂₅, R₇ and R₈ are independentlyselected from the group consisting of O, S, CHR₂₆, CHR₂₆, NR₂₆, and N;wherein R₂₆ is H, F or C₁-C₄ alkyl; R₁₅ is selected from the groupconsisting of hydroxy, phosphonate, and

wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH and S; y and m are integersindependently ranging from 0 to 4; p and q are integers independentlyranging from 1 to 10; n is an integer ranging from 0 to 10; or apharmaceutically acceptable salt or tautomer thereof, with the provisothat W and Y are not both methylene.
 2. The compound of claim 1 whereinthe compound is represented by the formula:

wherein R₁₅ is selected from the group consisting of hydroxy,phosphonate, and

wherein X and R₁₂ are independently selected from the group consistingof O and S; R₂₃ and R₂₄ are independently selected from the groupconsisting of H, F and C₁-C₄ alkyl; or a pharmaceutically acceptablesalt or tautomer thereof.
 3. The compound of claim 2 wherein y is 0 or1; n is 1-10; Z is CH₂; and R₁₇ is H.
 4. The compound of claim 2 whereiny is 0 or 1; n is 0; Z is C₅-C₆ aryl or C₅-C₆ heteroaryl; R₁₆ isselected from the group consisting of C₅-C₁₂ alkyl C₂-C₁₂ alkenyl orC₅-C₁₂ alkoxy; and R₁₇ and R₂₃ are each H.
 5. The compound of claim 4wherein Z is C₅-C₆ aryl; R₂₄ is H; and R₂₁ is selected from the groupconsisting of C₁-C₄ alkyl, and (C₁-C₄ alkyl)OH.
 6. The compound of claim1 wherein the compound is represented by the formula:

wherein Z is aryl or heteroaryl; R₁₆ is selected from the groupconsisting of C₅-C₁₂ alkyl, C₅-C₁₂ alkenyl, C₅-C₁₂ alkynyl and C₅-C₁₂alkoxy; Y is selected from the group consisting of CHOH, CF₂, CFH,carbonyl, NH, 0 and S; W is CR₂₇R₂₈; wherein R₂₇ and R₂₈ areindependently selected from the group consisting of H, halo and hydroxy;R₂₁ is selected from the group consisting of C₁-C₆ alkyl, (C₁-C₄alkyl)OH and (C₁-C₄ alkyl)NH₂; R₂₃ is selected from the group consistingof H, F, CO₂H, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄ alkyl)NH₂; R₂₄is selected from the group consisting of H, F and PO₃H₂, or R₂₃ togetherwith R₂₄ and the carbon to which they are attached form a carbonylgroup; R₁₅ is selected from the group consisting of hydroxy,phosphonate, and

wherein X and R₁₂ are independently selected from the group consistingof O and S; y is an integer ranging from 0 to 4; or a pharmaceuticallyacceptable salt or tautomer thereof.
 7. The compound of claim 6 whereinR₂₃ and R₂₄ are both H; R₂₇ and R₂₈ are independently selected from thegroup consisting of H and F; Z is C₅-C₆ aryl or C₅-C₆ heteroaryl; R₂₁ isselected from the group consisting of OH, C₁-C₄ alkyl, and (C₁-C₃alkyl)OH; and y is 0 or
 1. 8. The compound of claim 6 wherein thecompound is represented by the formula:

wherein R₁₅ is selected from the group consisting of hydroxy,phosphonate, and

wherein X and R₁₂ are independently selected from the group consistingof O and S; R₂₁ is selected from the group consisting of C₁-C₃ alkyl and(C₁-C₄ alkyl)OH; R₂₃ is selected from the group consisting of H, F,C₁-C₃ alkyl and (C₁-C₄ alkyl)OH; or a pharmaceutically acceptable saltthereof.
 9. The compound of claim 8 wherein Y is selected from the groupconsisting of carbonyl, NH and O.
 10. The compound of claim 9 whereinR₁₅ is OH; and R₂₃ is selected from the group consisting of H, F andC₁-C₃ alkyl; or a pharmaceutically acceptable salt thereof.
 11. Thecompound of claim 1 wherein the compound is represented by the formula:

wherein R₁₁ is selected from the group consisting of C₅-C₁₂ alkyl,C₅-C₁₂ alkenyl and C₅-C₁₂ alkyiyl; R₇ and R₈ are independently selectedfrom the group consisting of O, S, CHR₂₆, CHR₂₆, NR₂₆, and N; whereinR₂₆ is H, F or C₁-C₄ alkyl; R₂₅ is N or CH; R₂ is NH₂; R₃ is selectedfrom the group consisting of H, C₁-C₄ alkyl, (C₁-C₄ alkyl)OH, and (C₁-C₄alkyl)NH₂; R₁₅ is selected from the group consisting of hydroxy,phosphonate, and

wherein X and R₁₂ is selected from the group consisting of O and S; R₂₃is selected from the group consisting of H, F, OH, C₁-C₄ alkyl, CO₂H andC₁-C₄ alkyl; R₂₄ is selected from the group consisting of H, F, C₁-C₄alkyl and PO₃H₂, or R₂₃ together with R₂₄ and the carbon to which theyare attached form a carbonyl group; and y and m are integersindependently ranging from 0 to 4; or a pharmaceutically acceptable saltor tautomer thereof.
 12. The compound of claim 11 wherein m is 0; y is 0or 1; R₂₅ is CH; R₂₃ is H or F; and R₂₄ is selected from the groupconsisting of H, F and C₁-C₄ alkyl.
 13. The compound of claim 11 whereinR₃ is selected from the group consisting of C₁-C₃ allyl and (C₁-C₄alkyl)OH.
 14. The compound of claim 12 or 13 wherein R₇ is NH; and X isO; or a pharmaceutically acceptable salt or tautomer thereof.
 15. Thecompound of claim 14 wherein y is 0; and R₁₅ is OH.
 16. The compound ofclaim 13 wherein the compound is represented by the formula:

wherein R₁₁ is C₅-C₁₈ alkyl or C₅-C₁₈ alkenyl; and R₈ is N; or apharmaceutically acceptable salt or tautomer thereof.
 17. The compoundof claim 16 wherein R₁₅ is selected from the group consisting of hydroxyand

wherein R₁₂ is O or S; or a pharmaceutically acceptable salt or tautomerthereof.
 18. The compound of claimi 17 wherein R₁₁ is C₅-C₉ alkyl; R₁₅is OH and R₃ is selected from the group consisting of CH₃, CH₂CH₃,CH₂OH, CH₂CH₂OH and CH₂CH₂CH₂OH.
 19. A composition comprising a compoundof claim 1, 2, 6, 8, 11 or 16 and a pharmaceutically acceptable carrier.20. A composition comprising a compound represented by the formula

wherein R₁₁ is C₅-C₁₈ alkyl or C₅-C₁₈ alkenyl; Q is selected from thegroup consisting of C₃-C₆ optionally substituted cycloalkyl, C₃-C₆optionally substituted heterocyclic, C₃-C₆ optionally substituted aryl,C₃-C₆ optionally substituted heteroaryl and —NH(CO)—; R₂ is selectedfrom the group consisting of H, C₁-C₄ alkyl and (C₁-C₄ alkyl)OH; R₂₃ isH or C₁-C₄ alkyl, and R₁₅ is selected from the group consisting ofhydroxy, phosphonate, and

wherein X and R₁₂ is selected from the group consisting of O and S; or apharmaceutically acceptable salt or tautomer thereof and apharmaceutically acceptable carrier.
 21. The composition of claim 20wherein Q is selected from the group consisting of


22. The composition of claim 21 wherein R₁₅ is selected from the groupconsisting of hydroxy and

wherein R₁₂ is O or S.
 23. The composition of claim 22 wherein Q isselected from the group consisting of

R₁₅ is OH; or a pharmaceutically acceptable salt or tautomer thereof.24. A method for modulating the activity of an S1P receptor, said methodcomprising the step of contacting said receptor with a compoundrepresented by the formula:

wherein W is CR₂₇R₂₈ or (CH₂)_(n)NH (CO); wherein R₂₇ and R₂₈ areindependently selected from the group consisting of H, halo and hydroxy;Y is selected from the group consisting of a bond, CR₉R₁₀, carbonyl, NH,O or S; wherein R₉ and R₁₀ are independently selected from the groupconsisting of H, halo, hydroxy and amino; Z is CH₂, aryl, halosubstituted aryl or heteroaryl; R₁₁ and R₁₆ are independentiy selectedfrom the group consisfing of C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl, C₅-C₁₂ alkoxy, (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀ (aryl)R₂₀, C₅-C₁₀(heteroaryl)R₂₀, C₅-C₁₀ (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalkyl)R₂₀; wherein R₂₀ is Hor C₁-C₁₀ alkyl; R₂₉ is H or halo; R₁₇ is selected from the groupconsisting of H, halo, NH₂, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkylamino,C₁-C₆ alkylcyano and C₁-C₆ alkylthio; R₂, and R₂₁ are both NH₂; R₃ isselected from the group consisting of H, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH,and (C₁-C₄ alkyl)NH₂; R₂₂ is selected from the group consisting of C₁-C₆alkyl, (C₁-C₄ alkyl)OH and (C₁-C₄ akyl)NH₂; R₂₃ is selected from thegroup consisting of H, F, CO₂H, OH, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, and(C₁-C₄ alkyl)NH₂; R₂₄ is selected from the group consisting of H, F andPO₃H₂, or R₂₃ together with R₂₄ and the carbon to which they areattached form a carbonyl group; R₂₅, R₇ and R₈ are independentlyselected from the group consisting of O, S, CHR₂₆, CHR₂₆, NR₂₆, and N;wherein R₂₆ is H, F or C₁-C₄ alkyl; R₁₅ is selected from the groupconsisting of hydroxy, phosphonate, and

wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH and S; y and m are integersindependently ranging from 0 to 4; p and q are integers independentlyranging from 1 to 10; n is an integer ranging from 0 to 10; or apharmaceutically acceptable salt or tautomer thereof, with the provisothat W and Y are not both methylene.
 25. A method of providingimmuno-modulation to a patient in need thereof, said method comprisingthe step of administering to said patient a composition comprising acompound represented by the formula:

wherein W is CR₂₇R₂₈ or (CH₂),NH (CO); wherein R₂₇ and R₂₈ areindependently selected from the group consisting of H, halo and hydroxy;Y is selected from the group consisting of a bond, CR₉R₁₀, carbonyl, NH,O or S; wherein R₉ and R₁₀ are independently selected from the groupconsisting of H, halo, hydroxy and amino; Z is CH₂, aryl, halosubstituted aryl or heteroaryl; R₁₁ and R₁₆ are independently selectedfrom the group consisting of C₂-C₁₈ alkyl, C₂-C₁₅ alkenyl, C₂-C₁₈alkynyl, C₅-C₁₈ alkoxy, (CH₂)_(p)O(CH₂)_(q), C₅-C₁₀(aryl)R₂₀, C₅-C₁₀O(eterOaryI)R₂₀, CS5-CO (cycloalkyl)R₂₀, C₅-C₁₀ alkoxy(aryl)R₂₀, C₅-C₁₀alkoxy(heteroaryl)R₂₀ and C₅-C₁₀ alkoxy(cycloalklyl)R₂o; wherein R₂₀ isH or C₁-C₁₀ alkyl; R₂₉ is H or halo; R₁₇ is selected from the groupconsisting of H, halo, NH₂, C₁-C₆ alk.yl, C₁-C₆ alkoxy, C₁-C₆alkylamino, C₁-C₆ alkylcyano and C₁-C₆ alkylthio; R₂, and R₂₁ are bothNH₂; R₃ is selected from the group consisting of H, C₁-C₆ alkyl, (C₁-C₄aIkyl)OH, and (C₁-C₄ alkyl)NH₂; R₂₂ is selected from the groupconsisting of C₁-C₆ alkyl, (C₁-C₄ alkyl)OH and (C₁-C₄ alkyl)NH₂; R₂₄ isselected from the group consisting of H, F and PO₃H₂, or R₂₃ togetherwith R₂₄ and the carbon to which they are attached form a carbonylgroup; R₂₅, R₇ and R₈ are independently selected from the groupconsisting of O, S, CHR₂₆, CHR₂₆, NR₂₆, and N; wherein R₂₆ is H, F orC₁-C₄ alkyl; R₁₅ is selected from the group consisting of hydroxy,phosphonate, and

wherein R₁₂ is selected from the group consisting of O, NH and S; X isselected from the group consisting of O, NH and S; y and m are integersindependently ranging from 0 to 4; p and q are integers independentlyranging from 1 to 10; n is an integer ranging from 0 to 10; or apharmaceutically acceptable salt or tautomer thereof, with the provisothat W and Y are not both methylene.
 26. The method of claim 25 furthercomprising the step of administering a second immuno-modulatory agentselected from the group consisting of cyclosporine, tacrolimus,rapamycin, azathioprine, and corticosteroids such as prednisolone andprednisone.
 27. The method of claim 25 wherein the compound has thegeneral formula:

wherein R₁₁ is selected from the group consisting of C₁-C₂₂ alkyl,C₂-C₂₂ alkenyl and C₂-C₂₂ alkynyl; R₃ is selected from the groupconsisting of NH₂, OH, C₁-C₆ alkyl, (C₁-C₄ alkyl)OH, —(C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)aryl(C₀-C₄ alkyl) and (C₁-C₄ alkyl)aryloxyaryl(C₀-C₄alkyl); R₈ is selected from the group consisting of O, S and N. R₁₅ isselected from the group consisting of hydroxy, phosphonate, and

wherein R₁₂ is selected from the group consisting of O, NH and S; and Xis selected from the group consisting of O, NH and S; or apharmaceutically acceptable salt or tautomer thereof.
 28. A method ofpromoting wound healing in a warm blooded vertebrate, said methodcomprising the step of administering a composition comprising a acompound of the general structure:

wherein R₁ is C₅-C₁₈ alkyl or C₅-C₁₈ alkenyl; Q is selected from thegroup consisting of C₃-C₆ optionally substituted cycloalkyl, C₃-C₆optionally substituted heterocyclic, C₃-C₆ optionally substituted aryl,C₃-C₆ optionally substituted heteroaryl and —NH(CO)—; R₂ is selectedfrom the group consisting of H, C₁-C₄ alkyl and (C₁-C₄ alkyl)OH; R₂₃ isH or C₁C₄ alkyl, and R₁₅ is selected from the group consisting ofhydroxy, phosphonate, and

wherein X and R₁₂ is selected from the group consisting of O and S; or apharmaceutically acceptable salt or tautomer thereof.
 29. The method ofclaim 28 wherein Q is selected from the group consisting of —NH(CO)—,

and R₁₅ is selected from the group consisting of hydroxy and

wherein R₁₂ is O or S.
 30. The method of claim 29 wherein Q is selectedfrom the group consisting of

and R₁₅ is OH; or a pharmaceutically acceptable salt or tautomerthereof.
 31. A method for treating a patient suffering from a diseaseassociated with abnormal cell growth, said method comprising the stepsof administering a a compound of the general structure:

wherein R₁₁ is located in the meta or para position and is selected fromthe group consisting of C₅-C₁₈ alkyl and C₅-C₁₈ alkenyl; Q is selectedfrom the group consisting of C₃-C₆ optionally substituted cycloalkyl,C₃-C₆ optionally substituted heterocyclic, C₃-C₆ optionally substitutedaryl C₃-C₆ optionally substituted heteroaryl and —NH(CO)—; R₂ isselected from the group consisting of H, C₁-C₄ alkyl and (C₁-C₄alkyl)OH; R₂₃ is H or C₁-C₄ alkyl, and R₁₅ is selected from the groupconsisting of hydroxy, phosphonate, and

wherein X and R₁₂ is selected from the group consisting of O and S; or apharmaceutically acceptable salt or tautomer thereof.
 32. The method ofclaim 31 wherein Q is selected from the group consisting of —NH(CO)—;

and R₁₅ is selected from the group consisting of hydroxy and

wherein R₁₂ is O or S.
 33. The method of claim 32 wherein Q is selectedfrom the group consisting of

R₁₅ is OH; or a pharmaceutically acceptable salt or tautomer thereof.